Students Name:

Year/Section:

San Beda College of Medicine

Program for Medical Microbiology
Worksheet on Diagnostic Microbiology
Pacifico Eric E. Calderon, M.D
This is your Worksheet 2 in Medical Microbiology. Its main objective is to help you apply concepts in
medical bacteriology in hypothetical clinical scenarios.
I am sending you the worksheet in doc format. You MAY do this by pair, but you will have to
submit individually. Save the file in pdf format the send it to my email on or before August 24,
2015, 11:59AM. Send to doctorcocoy@yahoo.com
Note: This will serve only as a reviewer for your next long exam. The questions here are NOT
necessarily the questions that I will ask. NEVER!!! ;)

Solve the following cases briefly but intelligently.

A 2/M from a remote village in Sorsogon experienced an upper respiratory infection 2 weeks prior to
hospital admission. Four days prior to admission anorexia and lethargy were noted. The patient was
seen in the emergency room 3 days prior to admission. At that time he had a fever of 39.9C.
Physical examination revealed a clear chest, exudative pharyngitis, and bilaterally enlarged cervical
lymph nodes. A throat culture was taken and the child given a course of penicillin. On day 10 of the
infection, the child's condition worsened. He became increasingly lethargic; he developed respiratory
distress on the day of admission. It was noted that the throat culture from 3 days prior to admission
had not grown any group A streptococci. On examination the patient was febrile to 38.9 oC and had
an exudate in the posterior pharynx that was described as grayish, thick membrane which bled
when scraped and removed. The patients medical history revealed that he had received no
immunizations. The patient was admitted to hospital and treatment was begun.
What is the most likely diagnosis?
What is the most likely etiology?
How is this infection treated?

Diptheria
Corynebacterium diptheriae

Diphtheria antitoxin is given as a shot into a muscle or through an IV (intravenous line). The
infection is then treated with antibiotics, such aspenicillin and erythromycin. You may
need to stay in the hospital while getting the antitoxin.

How could this infection have been prevented?

People with diphtheria need to be kept in isolation until they are certified to be free of the
disease . Widespread immunization against diphtheria is the only effective control.

What virulence factors are responsible for the pseudomembrane formation on the throat? How are
these virulence factors acquired by the organism?
The diphtheritic lesion is often covered by a pseudomembrane composed of fibrin, bacteria, and inflammatory cells. Diphtheria toxin can
be proteolytically cleaved into two fragments: an N-terminal fragment A (catalytic domain), and fragment B (transmembrane and
receptor binding domains). Fragment A catalyzes the NAD+-dependent ADP-ribosylation of elongation factor 2, thereby inhibiting
protein synthesis in eukaryotic cells. Fragment B binds to the cell surface receptor and facilitates the delivery of fragment A to the
cytosol.

The detection of toxigenicity among Corynebacterium diphtheriae and Corynebacterium ulcerans

strains is the most important test for the microbiological diagnosis of diphtheria. How is toxigenicity
detected? What is this test called?
The detection of toxigenicity among Corynebacterium diphtheriae and Corynebacterium ulcerans strains is
the most important test for the microbiological diagnosis of diphtheria. Difficulties with current methods, in
particular the Elek test, are well documented. PCR may also be used for the detection of toxigenicity of
Corynebacterium species.

A 22/M army recruit presented to the clinic with a penile discharge. A Gram-stained preparation of
his urethral exudate revealed neutrophils with intracellular Gram-negative diplococci. The patient
was treated with ceftriaxone and doxycycline and was sent home. He was requested to return in one
week so that a urethral culture can be obtained to test for proof of antibiotic cure.
What is the most likely etiology?

Neisseria gonorrhoeae

Which culture medium should be used for the follow-up culture? Why?

Students Name:
Year/Section:

The current preferred laboratory method for the diagnosis of N gonorrhoeae infections is the isolation and
identification of the agent. The primary specimens should be inoculated onto nonselective chocolate agar
and selective agar containing antimicrobial agents that inhibit the growth of commensal bacteria and
fungi. The antibacterial agents in modified Thayer-Martin, Martin Lewis and New York City medium are
vancomycin, colistin, trimethoprim lactate and the antifungal agents nystatin and anisomycin or
amphotericin B. Some fastidious strains, such as the arginine-, hypoxanthine- and uracil-requiring strains,
are more susceptible to the concentrations of vancomycin or trimethoprim used in the selective media.
Isolates that are inhibited by supplements in selective media should be grown on media with lower
concentrations of antibiotic. Isolates that are atypical, such as vancomycin-susceptible strains, should be
forwarded to reference laboratories to confirm their identification. Therefore, a quality assessment
program that periodically compares isolation rates on selective and nonselective media is desirable.

Why was doxycycline added to his treatment regimen?

Doxycycline Capsules are used in the treatment of a variety of infections caused by susceptible strains of
Gram-positive and Gram-negative bacteria and certain other micro-organisms. Sexually transmitted
diseasessuch as Infections due to Chlamydia trachomatis including uncomplicated urethral, endocervical or
rectal infections. Non-gonococcal, urethritis caused by Ureaplasma urealyticum. Chancroid infections due to
Calymmatobacterium granulomatis. Alternative drug in the treatment of gonorrhoea and syphilis.

What is the likelihood (based on epidemiology) that this patient has an accompanying chlamydial
infection? Was Chlamydia trachomatis demonstrated in the urethral smear? Why?
How is Chlamydia trachomatis cultured?
Chlamydia species are obligate intracellular bacteria that require growth inside mammalian cells for propagation and survival.
As a result, Chlamydia cannot be grown on conventional bacteriological medium. This property makes Chlamydia difficult
organisms to grow and maintain in the laboratory. Up until 1965, passage in the yolk sac of the embryonated hen egg was the
only way to isolate and propagate the organism. Since then, a tissue culture system has been available that allows easier
laboratory culture of the Chlamydia species. However, with the exception of the LGV serovars, most C. trachomatis strains do
not readily infect tissue culture cells. Chemical or mechanical assistance is used to increase their infectivity. Today, large
numbers of infectious organisms can be purified through Renografin density gradient centrifugation of infected cell lysates.
The ability to propagate C. trachomatis in the laboratory has greatly increased the understanding of the pathogenesis of C.

A 43/M shepherd from Bukidnon developed a wound on he volar surface of his right arm, which later
progressed to a black, necrotic eschar with central ulceration. He tends cattle and goats in a
hacienda in Bukidnon.
What is the most likely etiology of this infection?

Bacillus
Anthracis

How will you confirm your diagnosis?

The differential diagnosis of a patient (farmer) with fever, adenopathy, and black eschar include other
cutaneous lesions such as furuncles (staphylococci), ecthyma gangrenosum (Pseudomonas aeruginosa), and
spider bites. However, none of these etiologies are known to cause eschar formation with surrounding edema.
The specific diagnosis of anthrax is made by growth of the organism from blood (inhalation anthrax), or wound
(cutaneous anthrax). Careful review of a Gram stain from a primary specimen of a patient with suspected
anthrax is necessary, because the organisms have the propensity to easily decolorize and appear gramnegative. However, the presence of spores is a key to the identification of the organism as a gram-positive
bacillus. Based on these few tests (large gram-positive bacilli, nonhemolytic, lecithinase positive) a
presumptive identification of Bacillus anthracis can be made. As a result of the recent events in the world
leading to concerns over bioterrorism, definitive diagnosis of anthrax must be performed in a public health
laboratory. Confirmatory testing involves the use of fluorescently labeled monoclonal antibodies as well as
DNA amplification assays. The use of India ink can also help to determine the presence of a capsule, a

What virulence factors involved in the pathogenesis of this disease?

Capsular polypeptide and anthrax

How is this disease treated?

Ciprofloxacin is the drug of choice for anthrax, following the identification of weaponized strains that were resistant to penicillin as a
result of the production of a -lactamase. Prevention of anthrax involves vaccination of animals as well as humans at high risk of
exposure (military personnel). Prophylaxis is not recommended for asymptomatic persons. When deemed necessary, prophylaxis with
ciprofloxacin must be maintained for up to 30 days because of the potential delay in germination of inhaled spores.

Currently, what is the role/value of Pasteurs vaccine in preventing this infection?

A 34/M blind man from Africa was noted to have corneal and scleral. You suspect that he went blind
because of a chlamydial disease.
What is the most likely diagnosis?

Trachom

Students Name:
Year/Section:
What is the most likely etiology? Give the specific strains involved in this infection.
Chlamydia trachomatis, an obligate intracellular human pathogen, C. trachomatis is a gramnegative bacterium, therefore its cell wall components retain the counter-stain safranin and appear pink under
a light microscope. It is ovoid in shape. C. trachomatis includes three human biovars:
serovars

Ab, B, Ba, or C cause trachoma: infection of the eyes, which can lead to blindness

serovars D-K cause urethritis, pelvic inflammatory disease, ectopic pregnancy,

What are Halberstaedter-Prowazek inclusions? How do they appear on examination of conjunctival

scrapings?
Inclusion bodies found in clusters in the cytoplasm of epithelial cells of the conjunctiva observed in the
infectious disease trachoma, caused by Chlamydia trachomatis.
Distinctive, complex, intracytoplasmic forms found in the conjunctival epithelial cells of people in the a
cute phase of trachoma, less frequently in later stages, varying from 1)discrete acidophilic granules (a
pproximately 250 nm in diameter), to 2) irregular clumps of such material embedded in a basophilic m
atrix, to 3) relatively large basophilicbodies (approximately 700-1000 nm in diameter), to 4) large baso
philic bodies that include discrete, tiny, acidophilic granules.

How is this disease transmitted?

Sexually transmitted and is the leading cause of infectious

How is this disease prevented?

Soft cheese imported from Mexico was implicated as the vehicle in an outbreak of meningitis and
bacteremia that occurred among attendees of a large company picnic. Twenty of the 40 affected
individuals were pregnant women. One of the victims had AIDS. Gram-positive, non-spore forming
rods were isolated from the cheese that had been stored in the cold.
What is the most likely cause of this outbreak?
How could this outbreak have been prevented? Why was Mexican cheese implicated in this
outbreak?
How is this organism cultured?
What is meant by tumbling motility in saline suspension?
Why is this organism called a facultative intracellular organism? What virulence factors are
responsible for this feature?
What is the role of cephalosporins in treating this infection? Explain briefly.