134 SEC TION II MICROBIOLOGY `M̀ICROBIOLOGY—CLINICAL BACTERIOLOGY

MICROBIOLOGY—CLINICAL BACTERIOLOGY
``

Gram-positive lab algorithm

Gram (purple/blue)

Branching
Bacilli Cocci
filaments

Aerobic Anaerobic Aerobic Anaerobic

Listeria Clostridium Nocardia Actinomyces

Bacillus Cutibacterium (weakly acid fast) (not acid fast)
Corynebacterium
(formerly
Propionibacterium)
Catalase

(Pairs or Streptococcus (Clusters) Staphylococcus

chains)
Hemolysis Coagulase

γ
(Partial (Complete
α hemolysis,
green)
β hemolysis,
clear)
(No hemolysis,
grows in bile)
S aureus
Novobiocin
Optochin sensitivity Bacitracin sensitivity Growth in 6.5% NaCl sensitivity
and bile solubility and PYR status and PYR Status

Group B Group A S saprophyticus S epidermidis

S agalactiae S pyogenes

Viridans streptococci
(no capsule) Enterococcus
S pneumoniae Nonenterococcus E faecium
S mutans (encapsulated) S bovis
S mitis E faecalis

Important tests are in bold. Important pathogens are in bold italics.

Note: Enterococcus is either α- or γ-hemolytic.

Gram-positive cocci antibiotic tests

Staphylococci Novobiocin—Saprophyticus is Resistant; On the office’s “staph” retreat, there was
Epidermidis is Sensitive no stress
Streptococci Optochin—Viridans is Resistant; Pneumoniae is OVRPS (overpass)
Sensitive
Bacitracin—group B strep are Resistant; group B-BRAS
A strep are Sensitive

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α-hemolytic bacteria Gram ⊕ cocci. Partial oxidation of hemoglobin causes greenish or brownish color without clearing
A around growth on blood agar A . Include the following organisms:
ƒ Streptococcus pneumoniae (catalase ⊝ and optochin sensitive)
ƒ Viridans streptococci (catalase ⊝ and optochin resistant)

β-hemolytic bacteria Gram ⊕ cocci. Complete lysis of RBCs  pale/clear area surrounding colony on blood agar A .
A Include the following organisms:
ƒ Staphylococcus aureus (catalase and coagulase ⊕)
ƒ Streptococcus pyogenes—group A strep (catalase ⊝ and bacitracin sensitive)
ƒ Streptococcus agalactiae—group B strep (catalase ⊝ and bacitracin resistant)

Staphylococcus aureus Gram ⊕, β-hemolytic, catalase ⊕, coagulase TSST-1 is a superantigen that binds to MHC
⊕ cocci in clusters A . Protein A (virulence II and T-cell receptor, resulting in polyclonal
A
factor) binds Fc-IgG, inhibiting complement T-cell activation and cytokine release.
activation and phagocytosis. Commonly Staphylococcal toxic shock syndrome (TSS)—
colonizes the nares, ears, axilla, and groin. fever, vomiting, rash, desquamation, shock,
Causes: end-organ failure. TSS results in  AST,  ALT,
ƒ Inflammatory disease—skin infections,  bilirubin. Associated with prolonged use of
organ abscesses, pneumonia (often after vaginal tampons or nasal packing.
influenza virus infection), endocarditis, Compare with Streptococcus pyogenes TSS (a
septic arthritis, and osteomyelitis. toxic shock–like syndrome associated with
ƒ Toxin-mediated disease—toxic shock painful skin infection).
syndrome (TSST-1), scalded skin syndrome S aureus food poisoning due to ingestion of
(exfoliative toxin), rapid-onset food preformed toxin short incubation period
poisoning (enterotoxins). (2–6 hr) followed by nonbloody diarrhea
MRSA (methicillin-resistant S aureus)— and emesis. Enterotoxin is heat stable not
important cause of serious nosocomial and destroyed by cooking.
community-acquired infections; resistance S aureus makes coagulase and toxins. Forms
due to altered penicillin-binding protein. fibrin clot around itself abscess.
mecA gene from staphylococcal chromosomal
cassette involved in penicillin resistance.

Staphylococcus Gram ⊕, catalase ⊕, coagulase ⊝, urease ⊕ cocci in clusters. Novobiocin sensitive. Does not
epidermidis ferment mannitol (vs S aureus).
Normal flora of skin; contaminates blood cultures.
Infects prosthetic devices (eg, hip implant, heart valve) and IV catheters by producing adherent
biofilms.

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Staphylococcus Gram ⊕, catalase ⊕, coagulase ⊝, urease ⊕ cocci in clusters. Novobiocin resistant.

saprophyticus Normal flora of female genital tract and perineum.
Second most common cause of uncomplicated UTI in young women (most common is E coli).

Streptococcus Gram ⊕, α-hemolytic, lancet-shaped Pneumococcus is associated with “rusty”

pneumoniae diplococci A . sputum, sepsis in patients with sickle cell
A Encapsulated. IgA protease. Optochin disease, and asplenic patients.
sensitive and bile soluble. Most commonly No virulence without capsule.
causes:
ƒ Meningitis
ƒ Otitis media (in children)
ƒ Pneumonia
ƒ Sinusitis

Viridans group Gram ⊕, α-hemolytic cocci. Optochin resistant Viridans group strep live in the mouth, because
streptococci and bile insoluble. Normal flora of the they are not afraid of-the-chin (op-to-chin
oropharynx. resistant).
Streptococcus mutans and S mitis cause dental Sanguinis = blood. Think, “there is lots of
caries. blood in the heart” (endocarditis).
S sanguinis makes dextrans that bind to fibrin-
platelet aggregates on damaged heart valves,
causing subacute bacterial endocarditis.

Streptococcus Gram ⊕ cocci in chains A . Group A strep “Ph”yogenes pharyngitis can result in
pyogenes (group A cause: rheumatic “phever” and glomerulonephritis.
streptococci) ƒ Pyogenic—pharyngitis, cellulitis, impetigo Strains causing impetigo can induce
A (“honey-crusted” lesions), erysipelas glomerulonephritis.
ƒ Toxigenic—scarlet fever, toxic shock–like Scarlet fever—blanching, sandpaper-like body
syndrome, necrotizing fasciitis rash, strawberry tongue, and circumoral
ƒ Immunologic—rheumatic fever, pallor in the setting of group A streptococcal
glomerulonephritis pharyngitis (erythrogenic toxin ⊕).
Bacitracin sensitive, β-hemolytic, pyrrolidonyl
arylamidase (PYR) ⊕. Hyaluronic acid capsule
and M protein inhibit phagocytosis. Antibodies
to M protein enhance host defenses against S
pyogenes but can give rise to rheumatic fever.
ASO titer or anti-DNase B antibodies indicate
recent S pyogenes infection.

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Streptococcus Gram ⊕ cocci, bacitracin resistant, β-hemolytic, Group B for Babies!

agalactiae (group B colonizes vagina; causes pneumonia,
streptococci) meningitis, and sepsis, mainly in babies.
Produces CAMP factor, which enlarges the
area of hemolysis formed by S aureus. (Note:
CAMP stands for the authors of the test, not
cyclic AMP.) Hippurate test ⊕. PYR ⊝.
Screen pregnant women at 35–37 weeks of
gestation with rectal and vaginal swabs.
Patients with ⊕ culture receive intrapartum
penicillin/ampicillin prophylaxis.

Streptococcus bovis Gram ⊕ cocci, colonizes the gut. S gallolyticus Bovis in the blood = cancer in the colon.
(S bovis biotype 1) can cause bacteremia and
subacute endocarditis and is associated with
colon cancer.

Enterococci Gram ⊕ cocci. Enterococci (E faecalis and Enterococci are more resilient than
E faecium) are normal colonic flora that are streptococci, can grow in 6.5% NaCl and bile
penicillin G resistant and cause UTI, biliary (lab test).
tract infections, and subacute endocarditis Entero = intestine, faecalis = feces, strepto =
(following GI/GU procedures). Catalase ⊝, twisted (chains), coccus = berry.
PYR ⊕, variable hemolysis.
VRE (vancomycin-resistant enterococci) are an
important cause of nosocomial infection.

Bacillus anthracis Gram ⊕, spore-forming rod that produces anthrax toxin (an exotoxin consisting of protective
antigen, lethal factor, and edema factor). Has a polypeptide capsule (poly d-glutamate). Colonies
show a halo of projections, sometimes referred to as “medusa head” appearance.
Cutaneous anthrax Painless papule surrounded by vesicles  ulcer with black eschar A (painless, necrotic)
A  uncommonly progresses to bacteremia and death.

Pulmonary anthrax Inhalation of spores, most commonly from contaminated animals or animal products, although
also a potential bioweapon  flu-like symptoms that rapidly progress to fever, pulmonary
hemorrhage, mediastinitis (CXR may show widened mediastinum), and shock. Also called
woolsorter’s disease.

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Bacillus cereus Gram ⊕ rod. Causes food poisoning. Spores survive cooking rice (reheated rice syndrome).
Keeping rice warm results in germination of spores and enterotoxin formation.
Emetic type causes nausea and vomiting within 1–5 hours. Caused by cereulide, a preformed toxin.
Diarrheal type causes watery, nonbloody diarrhea and GI pain within 8–18 hours.
Management: supportive care (antibiotics are ineffective against toxins).

Clostridia Gram ⊕, spore-forming, obligate anaerobic rods. Tetanus toxin and botulinum toxin are proteases
that cleave SNARE proteins involved in neurotransmission.
Clostridium tetani Produces tetanospasmin, an exotoxin causing Tetanus is tetanic paralysis.
tetanus. Tetanospasmin blocks release of
GABA and glycine from Renshaw cells in
spinal cord.
Causes spastic paralysis, trismus (lockjaw), risus
sardonicus (raised eyebrows and open grin),
opisthotonos (spasms of spinal extensors).
Prevent with tetanus vaccine. Treat with
antitoxin +/− vaccine booster, antibiotics,
diazepam (for muscle spasms), and wound
debridement.
Clostridium botulinum Produces a heat-labile toxin that inhibits Symptoms of botulism (the 4 D’s): Diplopia,
ACh release at the neuromuscular junction, Dysarthria, Dysphagia, Dyspnea.
causing botulism. In adults, disease is caused Botulinum is from bad bottles of food, juice, and
by ingestion of preformed toxin. In babies, honey (causes a descending flaccid paralysis).
ingestion of spores (eg, in honey) leads to Local botulinum toxin A (Botox) injections used
disease (floppy baby syndrome). Treat with to treat focal dystonia, hyperhidrosis, muscle
human botulinum immunoglobulin. spasms, and cosmetic reduction of facial
wrinkles.
Clostridium Produces α-toxin (lecithinase, a phospholipase) Perfringens perforates a gangrenous leg.
perfringens that can cause myonecrosis (gas gangrene A ;
A
presents as soft tissue crepitus) and hemolysis.
If heavily spore-contaminated food is cooked
but left standing too long at < 60°C, spores
germinate  vegetative bacteria  heat-labile
enterotoxin  food poisoning symptoms in
10-12 hours, resolution in 24 hours.

Clostridioides difficile Produces toxins A and B, which damage Difficile causes diarrhea.
B
enterocytes. Both toxins lead to watery diarrhea Treatment: oral vancomycin, metronidazole,
 pseudomembranous colitis B . Often 2° or fidaxomicin. For recurrent cases, consider
to antibiotic use, especially clindamycin or repeating prior regimen or fecal microbiota
ampicillin; associated with PPIs. transplant.
Diagnosed by PCR or antigen detection of one
or both toxins in stool.
Complications: toxic megacolon.

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Corynebacterium Gram ⊕ rods occurring in angular Coryne = club shaped (metachromatic granules
diphtheriae arrangements; transmitted via respiratory on Löffler media).
A droplets. Causes diphtheria via exotoxin Black colonies on cystine-tellurite agar.
encoded by β-prophage. Potent exotoxin ABCDEFG:
inhibits protein synthesis via ADP-ribosylation ADP-ribosylation
of EF-2, leading to possible necrosis in β-prophage
pharynx, cardiac, and CNS tissue. Corynebacterium
Symptoms include pseudomembranous Diphtheriae
pharyngitis (grayish-white membrane A ) with Elongation Factor 2
lymphadenopathy. Toxin dissemination may Granules
cause myocarditis, arrhythmias, neuropathies. Treatment: antibiotic therapy +/– diphtheria
Lab diagnosis based on gram ⊕ rods with antitoxin.
metachromatic (blue and red) granules and
⊕ Elek test for toxin.
Toxoid vaccine prevents diphtheria.

Listeria Gram ⊕, facultative intracellular rod; acquired by ingestion of unpasteurized dairy products and
monocytogenes cold deli meats, transplacental transmission, by vaginal transmission during birth. Grows well at
A refrigeration temperatures (“cold enrichment”).
Forms “rocket tails” (red in A ) via actin polymerization that allow intracellular movement and cell-
to-cell spread across cell membranes, thereby avoiding antibody. Characteristic tumbling motility
in broth.
Can cause amnionitis, septicemia, and spontaneous abortion in pregnant women; granulomatosis
infantiseptica; meningitis in immunocompromised patients, neonates, and older adults; mild, self-
limited gastroenteritis in healthy individuals.
Treatment: ampicillin.

Nocardia vs Both are gram ⊕ and form long, branching filaments resembling fungi.
Actinomyces Nocardia Actinomyces
A Aerobe Anaerobe
Acid fast (weak) A Not acid fast B
Found in soil Normal oral, reproductive, and GI flora
Causes pulmonary infections in Causes oral/facial abscesses that drain through
immunocompromised (can mimic TB but sinus tracts; often associated with dental caries/
with ⊝ PPD); cutaneous infections after extraction and other maxillofacial trauma;
trauma in immunocompetent; can spread to forms yellow “sulfur granules”; can also cause
B
CNS PID with IUDs
Treat with sulfonamides (TMP-SMX) Treat with penicillin
Treatment is a SNAP: Sulfonamides—Nocardia; Actinomyces—Penicillin

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Mycobacteria Gram ⊕ acid fast rods (pink rods, arrows in A ). TB symptoms include fever, night sweats,
A Mycobacterium tuberculosis (TB, often weight loss, cough (nonproductive or
resistant to multiple drugs). productive), hemoptysis.
M avium–intracellulare (causes disseminated, Cord factor creates a “serpentine cord”
non-TB disease in AIDS; often resistant appearance in virulent M tuberculosis strains;
to multiple drugs). Prophylaxis with activates macrophages (promoting granuloma
azithromycin when CD4+ count < 50 cells/ formation) and induces release of TNF-α.
mm3. Sulfatides (surface glycolipids) inhibit
M scrofulaceum (cervical lymphadenitis in phagolysosomal fusion.
children).
M marinum (hand infection in aquarium
handlers).

Tuberculosis
Mycobacterium Interferon-γ release assay (IGRA) has fewer
tuberculosis
Hilar nodes
false positives from BCG vaccination.
+ PPD ⊕ if current infection or past exposure.
Ghon
complex Ghon focus PPD ⊝ if no infection and in sarcoidosis or
(usually mid/ HIV infection (especially with low CD4+ cell
lower lobes)
Primary tuberculosis count).
> 90% < 10% Caseating granulomas with central necrosis
Healing by fibrosis Progressive primary tuberculosis and Langhans giant cell (single example
Calcification (AIDS, malnutrition)
(PPD )
in  A ) are characteristic of 2° tuberculosis.
Reactivation Progressive A
lung disease
2° tuberculosis
Bacteremia
Fibrocaseous
cavitary lesion
(usually upper Meninges
lobes) Miliary
tuberculosis
Vertebrae
(Pott disease)
Localized destructive disease Lymph nodes
Cavity
Caseation Lungs
Caseation
Spleen
Scar
Liver Adrenal
gland
Joints and
long bones

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Leprosy Also called Hansen disease. Caused by Mycobacterium leprae, an acid-fast bacillus that likes cool
A
temperatures (infects skin and superficial nerves—“glove and stocking” loss of sensation A ) and
cannot be grown in vitro. Diagnosed via skin biopsy or tissue PCR. Reservoir in United States:
armadillos.
Leprosy has 2 forms (many cases fall temporarily between two extremes):
ƒ Lepromatous—presents diffusely over the skin, with Leonine (Lion-like) facies B , and is
communicable (high bacterial load); characterized by low cell-mediated immunity with a
largely Th2 response. Lepromatous form can be Lethal.
B
ƒ Tuberculoid—limited to a few hypoesthetic, hairless skin plaques; characterized by high cell-
mediated immunity with a largely Th1-type response and low bacterial load.
Treatment: dapsone and rifampin for tuberculoid form; clofazimine is added for lepromatous form.

Gram-negative lab algorithm

Gram (pink)

Diplococci Coccobacilli Curved rods

Aerobic Haemophilus influenzae Oxidase

Bordetella pertussis
Maltose acid detection Pasteurella
Brucella
Francisella tularensis Grows in 42°C Grows in alkaline media Produces urease
Acinetobacter baumannii
N gonorrhoeae Campylobacter jejuni Vibrio cholerae Helicobacter pylori
N meningitidis
Moraxella

Bacilli

Lactose fermentation

Oxidase Fast Slow

E coli
Citrobacter
Klebsiella
Pseudomonas Serratia
H2S production Enterobacter
on TSI agar

Shigella Salmonella
Yersiniaa Proteus

Important tests are in bold. Important pathogens are in bold italics.

a
Pleomorphic rod/coccobacillus

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