UNIVERSITY OF SANTO TOMAS

Faculty of Pharmacy - Medical Technology

A.Y. 2020-2021
1st Term

20 Macalisang, Era Dawn Mae V. MT 6320

3C-MT December 10, 2020

BACTERIOLOGY LECTURE
OTHER LEARNING ACTIVITY (OLA)
THIRD (3​rd​) SHIFTING

CASE #1: 50 points

A 4 year-old girl with a history of tumor resection followed by radiation and chemotherapy
resulting in remission, presented with a one month history of fever, headache, vomiting and
more recently, neck stiffness. Upon admission to the hospital and further tests were done, it was
found that there was tumor growth recurrence.

A blood culture and sensitivity test was performed. The blood culture turned positive after 36
hours. Gram stain from positive blood culture bottle showed gram-negative rods or coccobacilli
that are non-motile. The colonies that grew on chocolate agar medium incubated at 5% CO2
showed non-hemolytic, large, smooth, round and convex with an opaque, colorless or grey hue.
The Trypticase agar added with sheep’s blood and the MacConkey Agar both did not show any
sign of growth. The Medical Technologist performed a serotyping capsular agglutination test on
colonies and noted that there was no agglutination on all antisera from a to f. The colony was
positive both for catalase and oxidase. The Med Tech further performed another assay by
preparing a bacterial suspension from the Chocolate Agar plate and swabbed it into a 5 %
Blood Agar Plate with horse blood. Then a colony of S. aureus was streaked in a perpendicular
direction on the BAP. The following day, tiny colonies grew around the colonies of S. aureus in
the BAP.

1. List all tests performed to arrive at presumptive identification of the bacteria

and explain the concept behind the phenotypical results observed that
support the identification.

● Blood culture - ​Haemophilus influenzae needs both X and V factors to be

able to grow. The blood agar plate contains blood, but it is not lyzed, so the
X factor is only available. Thus, it develops on chocolate agar that contains
lysed blood consisting of both factors. However, it can appear as tiny
satellite colonies around the colonies of other bacteria like ​Staphylococcus
aureus​, ​Streptococcus pneumoniae​, and ​Neisseria spp. as these
organisms produce V factor as a byproduct of metabolism.

● Gram stain - ​Haemophilus influenzae is gram-negative coccobacilli that

sometimes appears in pairs.
 ● CHOC agar - Chocolate blood agar (CBA) is used for cultivating fastidious
organisms like ​Haemophilus influenzae​, when incubated at 35-37°C in a
5% CO2 atmosphere. The CHOC agar contains lysed RBC so when
heated, the hemoglobin releases hemin, the X factor required by fastidious
organisms. Additionally, There was no hemolysis seen on the agar
because ​H. influenzae​ is non-hemolytic.

● Trypticase agar added with sheep’s blood and the MacConkey Agar -
Haemophilus spp. are V factor dependent and do not grow on SBA. It is
because sheep’s blood contains enzymes like NADases that hydrolyze the
V factor. Additionally, ​Haemophilus influenzae is not a lactose fermenter
bacteria that’s why there is no sign of growth on MacConkey Agar.

● Agglutination Test - There was no agglutination on all antisera from a to f

because the organism is nontypable ​H. influenzae​, which is a
non-encapsulated strain.

● Catalase Test - It is catalase positive because the organism contains

catalase.

● Oxidase Test - ​Haemophilus influenza​e is an oxidase positive.

● Bacterial suspension on BAP with Horse Blood - When grown near

Staphylococcus aureus, H. influenzae exhibits the “satellite phenomenon”.
It occurs because colonies of ​S. aureus produces V factor as a byproduct
of metabolism, allowing the H. influenzae to develop. Since I’ve mentioned
earlier that BAP has only X factor available on it, the ​H. influenzae needs
NAD or V factor to be able to grow.

2. Provide the presumptive identification of the bacteria

The bacteria is ​H. influenzae because the characteristics mentioned are

exact with the organism’s description. It is a fastidious gram-negative bacilli that
requires both X factor and V factor to be able to grow. It also does not grow on
SBA because the RBCs are still intact, and the sheep RBCs contain enzymes
(NADases) that hydrolyze V factor. With that, CHOC agar is commonly used to
recover ​H. influenzae f​ rom clinical specimens in laboratories. Additionally, it is
non-hemolytic, catalase and oxidase positive, non-lactose fermenter, and
produces satellitism in the presence of ​S. aureus, S. pneumoniae ​or​ Neisseria​ spp.

3. Provide the qualitative interpretation (Susceptible, Intermediate, Resistant)

of the results of zones of inhibition using Haemophilus Test Medium Disk
Diffusion Method by referring to the attached CLSI guideline for the
following antibiotics:

● Ampicillin (10ug) = 16 mm
 - Resistant
● Amoxicillin-Clavulanate 20/10 ug = 27 mm
- Susceptible
● Ceftriaxone (30ug) = 29 mm
- Susceptible
● Cefuroxime (30ug) =14 mm
- Resistant
● Cefonicid (30ug) = 18 mm
- Intermediate

4. Provide the final result

Identification result:​ ​Haemophilus influenzae

Susceptibility result: ​Haemophilus influenzae ​is susceptible to Amoxicillin-Clavulanate
(20/10 ug) and Ceftriaxone (30ug).

CASE # 2: 25 points

After two weeks of treatment, the patient came back with similar symptoms. The attending
physician requested for repeat blood culture. The medical technologist again grew the
organism as positive from blood culture after 26 hours of incubation. The growth was once
again seen from the CHOC agar, no growth on Sheep Blood Agar and MacConkey.
Subsequently, the following differential and additional tests were performed: 5% horse
blood hemolysis, X and V factor challenge: The following result was obtained.

A subculture on trypticase agar with 5% horse blood was done, and no hemolysis around
the colonies was noted. A beta-lactamase test was also performed and the result obtained
was negative. The physician requested for MIC reporting. Broth dilution for AST test was
performed on a cation-adjusted Mueller-Hinton Broth and yielded an MIC of 16 ug/ml for
Ampicillin. Referring to the guideline set by the CLSI (attached as Annex A), she noticed
that there were only two organisms for which the AST method for disk diffusion and MIC
broth dilution for ​Haemophilus spp​. was standardized. The following antibiotic results were
also read from the AST:

Amoxicillin-Clavulanate = R
Ampicillin-Sulbactam = S
Cefaclor = R
Cefamandole = S
Cefuroxime = S
Piperacillin-Tazobactam = S

1. List all tests performed to arrive at presumptive identification of the bacteria

and explain the concept behind the phenotypical results observed. What
could have caused the difference in growth yield between the first and
second culture? Provide the presumptive identification. (5 points)

● Blood culture - Normally, fastidious organisms like ​Haemophilus influenzae

do not grow in blood culture agar unless there are other bacteria present
that lyse the red blood cells.

● CHOC agar - Growth is seen in CHOC agar because it is used for

cultivating fastidious organisms.

● Sheep Blood Agar and MacConkey - There is no growth seen in Sheep

Blood Agar because blood of sheep contains NADases that hydrolyze V
factor needed for the organism’s growth. In addition, ​Haemophilus
influenzae does not ferment lactose that’s why no growth is seen in
MacConkey.

● Subculture on trypticase agar with 5% horse blood - Horse blood is

preferred over sheep blood because the former does not contain inhibitors.
Additionally, horse blood provides both ​X- (hemin) and V-factors (NAD)
required for the growth of fastidious microorganisms ​However, there is no
hemolysis seen because ​H. influenzae is unable to hemolyze the horse
blood cells on the agar is nonhemolytic and will only grow in the hemolytic
zone of ​S. aureus on blood agar plates because ​S. aureus releases V
factor as a byproduct of metabolism when they grow

● Quadriplate media - The only growth seen on the media are on the sides
where both hemin and NAD are present, and on the quadrant with 5%
horse blood.

● Beta-lactamase test - Beta-lactamase test is negative because the

organism is not resistant to antibiotics under beta-lactam group including
penicillin group, cephalosporin group, carbapenem, and monobactam.
Therefore, the organism is classified as beta-lactamase negative, ampicillin
resistant (BLNAR) that has an ampicillin MIC of ≥4.0 μg/ml
 ● Broth dilution for AST test - The organism is resistant to Ampicillin that’s
why it yielded a MIC of 16 ug/ml.

2. What is the interpretive result of the Ampicillin antibiotic? Given this result,
do you agree with the obtained in-vitro susceptibility result? Give the final
susceptibility result (applying the rule set by CLSI in reporting the final
antibiotic reporting for this organism). (15 points)

The Ampicillin antibiotic is not effective as a treatment because ​H.

influenzae is resistant to it. Yes, I agree with the obtained in-vitro susceptibility
result because according to studies there is increased resistance to Ampicillin that’s
why it should not be used solely for treatment. Additionally, the yielded MIC is 16
ug/mL, which is resistant based on the guidelines given by CLSI for Antimicrobial
Susceptibility Testing if the MIC is ​≥4 ug/mL

3. Provide the final result: (5 points)

Identification result:​ The organism is ​Haemophilus influenzae​ because it requires both

hemin (X factor) and NAD (V factor) for growth. Specifically, it is
beta-lactamase negative, ampicillin resistant (BLNAR).

Susceptibility result: Since the strain of ​Haemophilus influenzae recovered is

beta-lactamase negative ampicillin-resistant (BLNAR), the
susceptibility result will be changed to resistant even though it was
initially identified as susceptible to particular drugs including
Ampicillin-Sulbactam, Cefamandole, Cefuroxime, and
Piperacillin-Tazobactam.

CASE # 3: 25 points

The patient’s profile who went in for consultation was recorded as follows: Male, 38 y/o,
with notable ruptured postules on the thigh, and suppurative buboes on the inguinal area.
A sample was collected from the ruptured postule one day before, placed in a sterile
container and was submitted to the lab the following day. Culture results showed no
growth (negative). Due to the symptoms observed, the doctor requested for another
culture, and this time the sample collection was done in the laboratory with direct plate
swabbing and incubated at 5% CO2. The following day, there were noted colonies on the
Chocolate Agar plate as small, flat, non-mucoid colonies. While attempting to harvest
colonies from the chocolate agar, the Medical Technologist noticed that they were difficult
to get using a loop and the whole colony moved to the surface of the agar. The gram stain
showed railroad-like arrangement of gram-negative rods. Further X and V factor challenge
test was performed and it was noted that the isolate grew well in the presence of X factor,
and no growth in the V factor strip.

1. List all phenotypical tests that can be performed to arrive at a

presumptive identification of the bacteria in this case. Explain the
 concept behind each phenotypical results observed. What could have
caused the difference in growth yield between the first and second
culture? (15 points)

● Culture - Fastidious organisms won’t grow on normal culture.

● CHOC Agar - ​H. ducreyi is a fastidious organism that’s why it needs a

special medium like CHOC agar for development.

● Gram-stain - ​H. ducreyi displays pale staining gram-negative

coccobacilli. It is arranged singly or in clusters and commonly referred
to as “school of fish” or “railroad tracks”. Furthermore, it is loosely coiled
clusters of organisms lined up in parallel or appearing as “fingerprints.”

● X and V factor challenge test - ​H. ducreyi only requires X factor and
does not need V factor for growth unlike other Haemophilus spp. such
as ​H. influenzae​ and ​H. haemolyticus​.

2. With all the profile listed and their results obtained, correlate all
phenotypic output and provide the final result as follows: (10 points)

● Culture - The culture is negative because fastidious organisms only grow on

CHOC agar or where there needs for growth are met.

● CHOC Agar - The CHOC agar is positive because it is a special medium

used for the development and growth of fastidious organisms like
Haemophilus​ spp.

● Gram-stain - ​H. ducreyi ​is Gram-negative ​coccobacilli arranged singly or in

groups like “school of fish” or “railroad tracks”.

● X and V factor challenge test - ​H. ducreyi only requires X factor for growth
that’s why it didn’t grow in the V factor strip.

3. Presumptive Identification result: 5 points

The organism is Haemophilus ducreyi because when it was subjected to X and V

factor challenge test, the isolate grew well in the presence of X factor, and no growth in
the V factor strip.

Proposed antibiotic for this bacteria: 5 points

According to the Centers for Disease Control and Prevention (CDC), the
recommended first-line chancroid therapy is one of four regimens: azithromycin 1 g orally
in a single dose, ceftriaxone 250 mg intramuscularly in a single dose, ciprofloxacin 500
mg orally twice daily for 3 days, or erythromycin 500 mg orally 3 times daily for 7 days.
References:

Chocolate Agar. (2014, July 23). Retrieved December 2, 2020, from

https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1300.pdf

Haemophilus ducreyi (Chancroid) - Infectious Disease and Antimicrobial Agents. (n.d.).

Retrieved February 9, 2020, from
http://www.antimicrobe.org/new/b80.asp#:%7E:text=The%20recommended%20first%20
line%20therapy,day%20for%203%20days%2C%20or

Lablogatory, A. (2016, March 2). Microbiology Case Study: 6 Year Old Girl with Headache.
Retrieved February 9, 2020, from
https://labmedicineblog.com/2016/03/10/microbiology-case-study-6-year-old-girl-with-he
adache/

Musher DM. Haemophilus Species. In: Baron S, editor. Medical Microbiology. 4th edition.
Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 30.
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Tankeshwar, A. (2020, May 10). Chocolate Agar: Composition, uses and colony
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