Bacteriology Comp Exam

How does the age of bacterial cells affect Gram Stain results?

• Age

  • Old cells die, breaking down the peptidoglycan layer, compromising cell’s ability to retain CV. May also stain Gram-variable

• Antibiotics

  • Antibiotics that break down the cell wall or inhibit cell wall synthesis compromise cell’s ability to retain CV

Endotoxins (Lipopolysaccharides, (LPS))

Toxic structural molecules that make up the outer membrane of Gram-negative bacteria

They are passively released into the environment or a host's bloodstream when the bacterial cell dies, ruptures, or undergoes mechanical damage

Exotoxins

Highly potent, soluble proteins secreted by living bacteria (and some fungi or algae) into their surrounding environment or injected directly into host cells.

Act as targeted virulence factors that disrupt cellular metabolism, destroy tissue, or trigger severe, systemic immune responses.

Enterotoxins

Poisonous proteins secreted by bacteria that specifically target the intestines = food poisoning

What is thioglycolate broth used for?

To determine the oxygen requirements of microorganisms and to cultivate fastidious, aerobic, and anaerobic bacteria. The broth naturally forms an oxygen gradient, with high concentrations at the top and zero oxygen at the bottom. This allows microbiologists to classify bacteria as obligate aerobes, obligate anaerobes, facultative anaerobes, or microaerophiles based on where they grow in the tube.

Explain the principle of the acid-fast stain procedure

Identifies bacteria with thick, waxy cell walls, containing mycolic acid (appears bright red, retaining carbol fuchsin; non acid-fast: blue)

How do Beta-lactams work? + Examples

Inhibit wall synthesis

• Penicillin: GP

• Cephalosporins: GP and GN

• Monobactam: GN aerobes

• Carbapenem: Broad spectrum

• Cephamycin: GN and anaerobes

How do aminoglycosides work?

Inhibit protein synthesis

Targets aerobic GNs

How do glycopeptides work?

Inhibit cell wall synthesis

Targets GP + MRSA

What mechanism of resistance does beta-lactamase have?

Enzyme inactivation

Breaks the beta-lactam ring of the antibiotic

Can be inhibited by: Clavulanic acid, Tazobactam, Sulbactam, and Avibactam

What mechanism of resistance does carbapenemase have?

Enzyme inactivation

Enzyme destroys carbapenems, making broad spectrum β-lactams ineffective

What are the AST standards?

• Use 4-5 colonies, emulsify to reach a 0.5 McFarland standard density

• Mueller-Hinton agar base

• pH 7.2-7.4

• 35C

• Ambient

• 16-18 horus

E-test

AST test

A plastic strip contains a gradient of antibiotic concentrations. The point where the ellipse of inhibition intersects the strip is the MIC.

Microdilution

AST test

Organism is exposed to serial two-fold dilutions of antibiotics in broth to determine the MIC.

What are the possible mechanisms of resistance?

• Enzyme inactivation

• Altered receptors

• Altered permeability

• Altered metabolic pathways

• Biofilm production

Draw the pathway for Staphylococcus/Micrococcus

Compare: morphology, novobiocin susceptibility, coagulase production (tube/latex)

• S. aureus

• S. epidermidis

• S. saprophyticus

• S. lugdunensis

• Micrococcus

 

	S. aureus	S. epidermidis	S. saprophyticus	S. lugdunensis	Micrococcus
Morph	cream/gold, beta-hemolytic	gray-white, nonhemolytic	white, creamy, nonhemolytic	very white, nonhemolytic	white/yellow/pink
Novobiocin	Susceptible	Susceptible	Resistant	Susceptible	N/A
Coagulase	Positive	Negative	Negative	Negative	Negative

What does S. aureus cause?

1) Skin infections

2) Pneumonia

3) Osteomyelitis

4) Wound infections

5) Toxin mediated illness

6) Opportunistic infections

What does S. epidermidis cause?

1) Subacute bacterial endocarditis (SBE)

2) Infections associated with prosthetic devices

What does S. saprophyticus cause?

UTIs (especially in young women)

What virulence factors does S. aureus have?

• Clumping factor

• Coagulase

• Fibrinolysin

• Hyaluronidase

• Beta-lactamase

• Exotoxins

• Protein A

What is clumping factor?

a VF: surface protein that binds fibrinogen, causing bacteria to clump

Helps adhesion and avoid phagocytosis

What is coagulase?

A VF: converts fibrinogen into fibrin, forming a clot

Protects bacteria from immune cells

What is fibrinolysin/staphylokinase?

A VF: breaks down fibrin clots

Allows bacteria to spread

What is hyaluronidase?

A VF: Breaks down hyaluronic acid in connective tissue

Allows bacteria to spread

What is Protein A?

A VF: binds the Fc portion of IgG antibodies

Prevents antibodies being recognized by phagocytes

What virulence factors does S. epidermidis have?

• PGA capsule

• Biofilm formation

What is modified oxidase (Microdase) test used for?

To differentiate Micrococcus (pos) from Staphylococcus (neg)

Describe the use of Mannitol Salt agar in the isolation of Staphylococcus aureus.

Staphylococcus thrive in salt, but S. aureus can ferment the mannitol (other Staph cannot)

How would you identify MRSA?

MRSA

• Resistance to penicillin, oxacillin, vancomycin, and cefoxitin

• Has mecA and PBP2a

• Use cefoxitin (30ug) and oxacillin (1ug) disk diffusion tets to predict mecA-mediated resistance

• Utilize AST standards

What does Group A (Strep pyogenes) cause?

1. Pharyngitis

2. Erysipelas

3. Impetigo

4. Scarlet fever

5. Necrotizing fasciitis

6. Toxic shock syndrome

7. Post-streptococcal sequelae (nonsuppurative complications)

   1. Acute glomerulonephritis

   2. Rheumatic fever

8. Wounds

9. Opportunistic infections

What does Group B (Strep agalactiae) cause?

1. Neonatal pneumonia

2. Neonatal meningitis

3. Postpartum infections

4. Urinary tract infections (UTI)

5. Osteomyelitis

What does Strep bovis group (Group D) cause?

Bacteremia associated with colon malignancy

What does Enterococcus cause?

1. Urinary tract infections (UTI)

2. Mixed infections of the abdomen and pelvis

3. Wounds

What does Viridans strep cause?

Subacute bacterial endocarditis

What does Strep pneumoniae cause?

1. Pneumonia

2. Meningitis

3. Otitis media

4. Sinusitis

Draw the Streptococcaceae flowchart

Hippurate Hydrolysis

detects ability to hydrolyze hippurate

Bile-esculin

ability to hydrolyze esculin in presence of bile

Bile solubility

bile activates enzymes that lyse susceptible bacteria

What virulence factors does Group A Strep have?

• Streptolysin O: oxygen-labile hemolysin, causes tissue damage

• Streptolysin S: oxygen-stable hemolysin, responsible for beta-hemolysis

• M protein: surface protein that prevents phagocytosis by inhibiting complement

What virulence factor(s) does Strep pnuemoniae have?

Capsule - prevents phagocytosis

For which organism should you perform AST?

1. Beta-hemolytic strep

2. Viridians strep

3. Strep pneumoniae

For virdians and strep pneumoniae

How do you set up culture for urine specimens?

For clean-catch/catheter: use 0.001 loop, streak for colony count, BAP and MAC, ambient, 35C

What are the expected test results from a BactiCard for Neisseria sp and Moraxella catarrhalis?

BactiCard Performed on isolates from enriched selective media only
	N. gonorrhoeae	N. meningitidis	N. lactamica	M. catarrhalis
IB Pos: teal	Negative	Negative	Negative	Positive
PRO Pos: purp	Positive	Variable	N/A	N/A
GLUT Pos: purp	Negative	Positive	N/A	N/A
BGAL Pos: teal	Negative	Negative	Positive	N/A

What is Thayer-Martin media used for?

CHOC agar with added antibiotics (Vancomycin, colistin, nystatin, trimethoprim)

Selective for pathogenic Neisseria sp.

• N. gonorrhoeae: small, gray to tan, translucent, raised

• N. meningitidis: small, tan, sometimes mucoid, convex

• N. lactamica (non-pathogenic, but still grows)

Culture requirements for Neisseriaceae

• Transported at room temp, susceptible to drying

• Capnophilic (2-8% CO₂)

N. gonorrhoeae

• Media: CHOC and modified Thayer-Martin

• Inoculate with dacron or rayon swabs

N. meningitidis

• Media: SBA, CHOC, modified Thayer-Martin

What does Neisseria gonorrhoeae cause?

1) Gonorrhea

2) Ophthalmia neonatorum

3) Pelvic inflammatory disease (PID)

4) Disseminated gonococcal infections (DGI)

What does Neisseria meningitidis cause?

1) Meningitis

2) Septicemia

What does Moraxella catarrhalis cause?

1) Otitis media/maxillary sinusitis in children

2) Pneumonia/bronchitis in immunocompromised

Describe the following characteristics common to the Enterobacterales

• GNR / GNCB

• Oxidase negative (except Plesiomonas)

• Glucose fermenters

• Nitrate reduction positive

• Growth on MAC + HE/XLD agar

Compare all of the GNR selective agars

Agar	Selects	Differentiates	Results	Notes
XLD (Red)	GNR	Xylose, lactose, sucrose fermenters  Lysine decarboxylation (LCD)  H2S producers	XF, no LCD = yellow XF, LCD = red H2S = black ppt	Lower bile salt concentration = better Shigella recovery
HE (Blue/ Green)	GNR	Lactose, sucrose fermenters  H2S producers	L/SF: yellow/orange NF: colorless b/g H2S: black ppt	Higher concentration of bile salts than MAC  Still inhibits many normal flora Enterics
SS (Pink)	GNR	Lactose fermenters  H2S producers	LF: pink NLF: colorless H2S: black ppt	Very inhibitory, but other Enterics can grow
SMAC (Pink)	GNR	Sorbitol fermenters	SorbitolF: pink SorNF: colorless
MAC (Pink)	GNR	Lactose fermenters	LF: pink NLF: colorless	Bile salt and CV
CIN/ Yersinia	GNR Y. entercolitica	Mannitol fermenters	MF: pink NMF: colorless	Yersinia have bulls-eye look  Ingredients inhibit most organisms found in stool
EMB	GNR	Lactose, sucrose fermenters	Fermenters: purple, strong F: metallic  NF: colorless

What is the purpose of using Gram-negative broth?

Selectively enriched to promote Salmonella and Shigella sp

Clinically significant lactose-fermenting Enterobacterales

• Escherichia coli

• Klebsiella pneumoniae

• Klebsiella oxytoca

• Klebsiella (Enterobacter) aerogenes

• Enterobacter cloacae

• Citrobacter freundii

Clinically significant non-lactose fermenting, LDA/TDA positive, ONPG negative Enterobacterales

• Proteus vulgaris

• Proteus mirabilis

• Morganella morganii

Clinically significant non-lactose fermenting, LDA/TDA negative Enterobacterales

• Shigella sonnei

• Shigella species

• Salmonella species

• Salmonella typhi

• Serratia species

• Citrobacter koseri

• Yersinia entercolitica

Draw out the abbreviated ID scheme for E. coli

Draw out the abbreviated ID scheme for Proteus sp

What is the only lactose fermenting GNR that also produces H₂S?

Citrobacter freundii

Define the following terms:

a. Capsular antigen (K)

b. Somatic antigen (O)

c. Flagellar antigen (H)

d. Vi antigen

These are specific surface structures used to classify and identify bacteria

• Capsular antigen (K): surrounds bacterial cell outside the O antigen, protection from phagocytosis and complement

• Somatic antigen (O): heat-stable polysaccharide portion of the lipopolysaccharide (LPS) in the outer membrane of GN bacteria

• Flagellar antigen (H): heat-labile protein (flagellin) that makes up flagella

• Vi antigen: surface capsular polysaccharide antigen that overlies O antigen, inhibiting phagocytosis

Which Enterics are always pathogens?

• Shigella sp

• Salmonella sp

• Yersinia

• E. coli O157:H7

What does E. coli cause?

• UTI

• Wound infections

• Hemolytic uremic syndrome (HUS)

• Neonatal meningitis

• GI infections

What do Salmonella/Shigella sp cause?

• GI infections

• Typhoid fever (Salmonella typhi)

What do Proteus sp cause?

• Wound infections

• UTI

• Opportunistic/nosocomial infections

What does K. pneumoniae cause?

• Opportunistic/nosocomial infections

• Pneumonia

• Wound infections

What does Yersinia cause?

• GI infections

Which Enterics are commonly responsible for opportunistic/nosocomial infections?

• K. pneumoniae

• Enterobacter cloacae

• Serratia sp

• Citrobacter sp

• Proteus sp

• Morganella morganni

Culture setup for Haemophilus

• CHOC (fastidious organisms)

• X (Hemin) and V (NAD) factor

• CO₂ (Capnophilic)

• Moist environment

• Incubation for 24-48 hours

Gram stain and colonial morphology of Haemophilus species.

• GNCB

• Tan, translucent, mousy odor

• H. ducreyi: small, grey, yellow/tan, hockey-puck

Describe the difference in test results between H. influenzae and H. parainfluenzae

(X/Y test; Porphyrin; Horse blood; Satellitism)

	Influenzae	Parainfluenzae
X/Y Test (Growth)	XV	V & XV
Porphyrin	Negative	Positive
Horse Blood	No	No
Satellitism	Positive	Positive

What is H. influenzae’s most significant virulence factor?

Capsule

• Encapsulated strains, especially Hib cause severe invasive infections, especially in unvaccinated children younger than 5

• Un-encapsulated species can still cause disease

  • Localized infections (elderly and immunocompromised)

What kind of diseases do H. influenzae cause?

• Respiratory infections

• Meningitis

• Otitis media

• Conjunctivitis (ssp. aegypticus

Fill in this table for Glucose NF GNRs

	Oxidase	42C	ADH	ODC	LDC	Gelatin	Polymyxin B
P. aeruginosa
S. maltophilia
B. cepacia

	Oxidase	42C	ADH	ODC	LDC	Gelatin	Polymyxin B
P. aeruginosa	+	Growth	+	-	-	+/-	Susceptible
S. maltophilia	-	No	-	-	+	+	Susceptible
B. cepacia	+/-	+/-	-	+/-	+	+/-	Resistant

What are the glucose non-fermenting GNRs?

• P. aeruginosa

• S. maltophilia

• B. cepacia

Why are P. aeruginosa and S. maltophilia not considered Enterobacterales?

They are:

• Oxidase positive

• Glucose utilizers, not fermenters

Compare the morphologies of P. aeruginosa vs. S. maltophilia

Characteristic	Pseudomonas aeruginosa	Stenotrophomonas maltophilia
Colony morphology	Large, flat, spreading colonies; may have irregular edges, metallic sheen, and beta-hemolysis	Smooth, glistening colonies that may appear yellow to lavender-green; usually nonhemolytic

Draw the flowchart for GNF GNR

GNF GNRs are primarily ____ pathogens

opportunistic

What kind of diseases can P. aeruginosa cause?

• Nosocomial infections

• Wounds, burns, respiratory

• Otitis externa

• Corneal ulcers

• UTI

• Cystic fibrosis

• Osteomyelitis

What kind of diseases can S. maltophilia cause?

• Respiratory

• Wound

• Nosocomial

What is the difference between culture set up for a throat vs sputum specimen?

SPUTUM

• BAP, CHOC, MAC

THROAT

• Strep selective blood agar (anaerobic)

• To find Group A strep only, streptolysin

Both require a direct gram stain, streak for isolation, 48 hours minimum

Pathogens vs normal flora of the respiratory tracts

Flora (upper)

• Viridans strep

• CoagN Staph

• Nonpathogenic Neisseria

• Diphtheroids

• Anaerobes

• Yeast

Pathogens (lower; normally sterile)

• S. pneumoniae

• H. influenzae

• M. catarrhalis

• K. pneumoniae

• E. coli

• Enterobacter spp

• Serratia marcescens,

• S. aureus

• P. aeruginosa

• S. maltophilia

• Acinetobacter sp

• S. pyogenes

• B. cepacia

Should you perform a gram stain or acridine orange stain on sterile body fluids/tissues?

• A gram stain should be performed ASAP, only to guide empiric treatment

• Acridine orange can be used when gram stain is negative, but infection is suspected or the specimen contains few organisms (supplemental to gram stain)

Describe the proper procedure for setting up each of the following cultures, including proper specimen preparation, types of media used, and incubation requirements:

• Cerebrospinal fluid

• Sterile body fluids

• Solid tissues

Specimen	Preparation	Media	Incubation
CSF	Process ASAP; never refrigerate. >1 mL: centrifuge; <1 mL: plate directly	BAP, CHOC, MAC, Thio	35°C; aerobic: 48 hr; Thio: 7 days
Sterile body fluids	Process ASAP. >1 mL: centrifuge; <1 mL: plate directly. Use anaerobic transport if needed	BAP, CHOC, MAC, Thio ± anaerobic media	35°C; aerobic: 48 hr; anaerobic: 72 hr; Thio: 7 days
Solid tissues	Keep moist; touch prep, then grind/homogenize. If fungi suspected: mince, do not grind	BAP, CHOC, MAC, Thio ± anaerobic media	35°C; aerobic: 48 hr; anaerobic: 72 hr; Thio: 7 days

Where are obligate anaerobes are found in the environment and the human body?

Location	Examples
Environment	Soil, deep water sediments, sewage, and decaying organic material
Body	Mouth, colon, vagina, and deep skin

Predisposing Conditions of Anaerobic Infections

Predisposing Conditions

• Trauma of mucosal membranes 

• Vascular stasis

• Tissue necrosis 

• Aspiration of oral contents into lungs 

• Decrease in redox potential of tissues

What types of specimens may be submitted for anaerobic cultures? What can you collect them in?

• Tissues

• Sterile body fluids

• Blood and bone marrow

• Suprapubic/Lung/Transtracheal aspirate

• Wound drainage

Collect with: 

• Anaerobic transport tubes 

• Biobag

• Syringe with cap

• Blood culture bottles

Process immediately in a BSC or anaerobic chamber

Signs of an anaerobic infection

• Purulent specimen 

• Gas, foul odors 

• Black exudate w/ possible brick red fluorescence

• Sulfur granules

• Necrosis or gangrene 

• Infections close to mucosal surfaces 

• Infection persists despite aminoglycoside therapy 

• Characteristics morphology: double zone of hemolysis, molar tooth/breadcrumb colony, ground glass/fried egg colony

What is the purpose of using ABAP?

• Isolate and grow obligate anaerobes

• Enriched with sheep blood, hemin, and vitamin K

What is the purpose of pre-reduced media?

Pre-reduced media have oxygen removed to grow anaerobes

Aerotolerance testing

Subculture a suspected anaerobic colony to BAP, CHOC, and AnaBAP to prove colony is true obligate anaerobe vs facultative anaerobe

 

CHOC	AnaBAP	Obligate Anaerobe
Yes	Yes	No
No	Yes	Yes

What is the most common anaerobic organism isolated in the clinical lab?

Bacteroides fragilis

What are examples of Gram-positive Bacilli? (GPR) + GS morphology, catalase reaction, oxygen requirements

• Bacillus sp - spore forming

• Corynebacterium sp - irregular, palisading

• Listeria monocytogenes - regular, small

All are catalase positive and facultative anaerobes

Compare GS, colony morphology, and key tests for different GPRs

Organism	Gram stain	Colony	Key tests
B. anthracis	Large GP rods with spores	Nonhemolytic	Nonmotile, catalase +
Other Bacillus	Large GP rods with spores	Usually beta-hemolytic	Usually motile, catalase +
Diphtheroids	Irregular GP rods; Chinese letters	Small, usually nonhemolytic	Nonmotile catalase +
L. monocytogenes	Small GP rods	Narrow beta-hemolysis	Motile hippurate + catalase +

What diseases does Listeria monocytogenes cause?

• Meningitis

• Listeriosis

Should you direct gram stain a wound culture?

Yes, correlate with culture

Describe the procedure for setting up a wound culture using the following specimens:

• Aerobic swab

• Aerobic swab and anaerobic transport system

• Aspiration

Inoculate with swab, streak for isolation

• Aerobic plates

  • Chocolate agar (CHOC)

  • Sheep blood agar (BAP)

  • PEA or CNA

  • MacConkey agar (MAC)

• Anaerobic plates

  • Supplemented anaerobic blood agar (ABAP)

  • Kanamycin, Vancomycin (KV) agar

  • Anaerobic PEA (APEA)

  • Thioglycollate broth (THIO)

Incubate media

• Temperature: 35ºC

• Atmosphere:

  • BAP, CHOC, CNA, PEA - CO2

  • MAC, THIO - ambient air

  • ABAP, KV agar, and Ana PEA - anaerobically

• Time:

  • Aerobic media: total 48 hours

  • Anaerobic: read at 48 hours, 72 hours total or until ID complete

  • Thioglycollate broth: typically 5 to 7 days or longer if serious infection suspected (2 weeks)

What is Campylobacter blood agar (CVA) or Skirrow blood agar used for?

• Selects for Campylobacter sp from stool

• Incubated under microaerophilic, capnophilic conditions at 42C for 48-72 hours

• Contains blood and antibiotics (cefoperazone, vancomycin, and amphotericin B) to inhibit other organisms

• Colony: Small, gray, moist colonies that may appear flat, spreading, or “water-droplet-like”

Describe identifying characteristics of Campylobacter jejuni and what diseases it causes

• Growth on CVA/Skirrow agar

• Microaerophilic, capnophilic

• Gram-negative thin, curved rods

• Only grows at 37C or 42C

• Gray, moist, flat, spreading

• Catalase: positive

• Oxidase: positive

• Sodium Hippurate: positive

• Motility: positive

Causes: gastroenteritis

What media should you use for a stool culture?

• Salmonella/Shigella species

• Campylobacter jejuni

• Escherichia coli O157:H7

• Yersinia enterocolitica

• Staphylococcus aureus

• Salmonella/Shigella species: MAC, HE/XLD

• Campylobacter jejuni: CVA/Skirrow (42C)

• Escherichia coli O157:H7: SMAC

• Yersinia enterocolitica: CIN (incubate at 25C)

• Staphylococcus aureus, Plesiomonas shigelloides, Aeromonas hydrophila : BAP