STREPTOCOCCUS, ENTEROCOCCUS, AND

OTHER CATALASE-NEGATIVE GRAM-POSITIVE COCCI

General Characteristics
Gram (+), Catalase (-) spherical cells in pairs or chains (grown in broth cultures)
Differentiates Strepto and Entero to Staphylo Negative Catalase Test
More elongated
Facultative anaerobes or aerotolerant anaerobes
Metabolism - lactic acid fermentation
Increased CO2 for growth (capnophilic)
Growth more pronounced in enriched agar
Small, transparent colonies (pinpoint)
Cell Wall Structure
Species

S. pyogenes
S. agalactiae
S. dysgalactiae, S. equi
S. bovis group
E. faecalis, E. faecium
S. pneumoniae
Anginosus, Mutans, Mitis,
Salivarius group

Lancefield
Group
Antigen
A
B
C
D
D
none
A, C, F, G, N,
or none

Hemolysis
Types

Common Terms

, none
, , none

Group A Strep
Group B Strep
Group C Strep
Members of Viridans strep
Enterococcus
Pneomococcus

, , none

Viridans Strep

Hemolysis
Hemolysis
Alpha ()

Description
partial lysis; greenish
coloration

Beta ()

complete lysis; clear area

Nonhemolytic or
Gamma

no lysis; no change in agar

Alpha-prime ()

small area of intact RBC;

wider zone of complete
hemolysis

Streptococcus pyogenes

Antigenic Structure
Lancefield group A
M protein
o Attached to the peptidoglycan of the cell wall
o Extends to the cell surface
o Essential for virulence (encoded by the emm genes)
Virulence Factors
More than 80 different serotypes of M protein
M protein molecule
o Streptococcal cells resist phagocytosis
o Adherence of the bacterial cell to mucosal cells
Additional virulence factors
o Lipoteichoic Acid and Protein F mediate adherence to host epithelial cells
o Hyaluronic Acid Capsule prevents opsonized phagocytosis; mask its antigens
and remain unrecognized
o Deoxyribonuclease (DNase) A, B, C and D; antigenic
o Hemolysins
Streptolysin O
- Hemolysis on sheep blood agar (SBA)
- Oxygen labile
- Active in the reduced form (anaerobic environment)
- Lyses leukocytes, platelets and other cells as well as RBC
- Highly immunogenic
- Antistreptolysin-O (ASO) test for recent infection
Streptolysin S
- Oxygen stable
- Lyses leukocytes
- Nonimmunogenic
- Hemolysis around the colony aerobic incubation
o Enzymes
Streptokinase lyses the fibrin clot
Hyaluronidase spreading factor/invasiveness
Streptodornase dissolve purulent or fibrinous secretions
o Erythrogenic Toxin scarlet fever (red spresding rash)
SpeA, SpeB and SpeC superantigens
- Highly mitogenic proteins
- Stimulate T-lymphocyte proliferation production of IL-1, tumor
necrotizing factor and other cytokines
Clinical Infections
Bacterial pharyngitis and tonsilitis
o Strep throat
o 1 -4 days of incubation period abrupt onset
Sore throat, malaise, fever and headache
Tonsils and pharynx are inflamed
Cervical lymph nodes are swollen and tender

o Spread by droplets and close contact

o Diagnosis: Throat culture or Direct Antigen Detection
Pyodermal Infections
o Impetigo (Young children 2-5 years)
Small vesicles weeping lesions crust over
Affects exposed areas of the skin
Inoculation through minor abrasions or insect bites
o Erysipelas (Elderly patients)
Acute spreading skin lesion
Erythematous with a plainly demarcated but irregular edge
o Cellulitis
Deeper invasion
Life threatening
With bacteremia or sepsis
Patients with peripheral vascular disease or diabetes gangrene
o Wound infection
o Arthritis
Scarlet Fever (Erythrogenic toxin Red rash)
o Rash disappear followed by desquamation
o Tongue yellow-white with red papillae (strawberry tongue)
o Diagnosis:
Dicks Test
- Susceptibility test for scarlet fever
- Erythrogenic toxin injected
- Positive redness and swelling
Schultz-Charlton Reaction
- Diagnostic test for scarlet fever
- Antierythrogenic toxin injected
- Rash will blanch then fade
Necrotizing Fasciitis
o Flesh-eating disease
o Rapidly progressing inflammation and necrosis of the skin, subcutaneous fat,
and fascia
o Life threatening
o Occurs to someone who has suffered trauma to the skin portal of entry
Streptococcal Toxic Shock Syndrome
o Entire organ system shuts down leading to death
Post-Streptococcal Sequelae
o Rheumatic Fever fever and inflammation of the heart, joints, blood vessels
and subcutaneous tissues
o Acute Glomerulonephritis damage to the glomeruli and impairment of
kidney function
o Treatment: Penicillin or Erythromycin (patients allergic to penicillin)

Laboratory Diagnosis
Proper sampling
o Tongue should be depressed and swab rubbed over the posterior pharynx
o If exudates is present, it should also be touched by the swab

Transport media are not required for normal conditions

o Organism is resistant to drying
o Can be recovered from swabs several hours after collection
SBA plate is inoculated and streaked for isolation
o SBA containing SXT recommended for better recovery of -hemolytic
streptococci from throat cultures
o Observed after 24 hours for the presence of -hemolytic colonies
o If none are found, additional 24-hour incubation before the culture is reported
negative
o Colonies: small, transparent and smooth w/ a well-defined are of -hemolysis
o Gram stain: gram (+) cocci, short chains
Key tests: Bacitracin susceptibility or PYR hydrolysis

Streptococcus agalactiae

Antigenic Structure
Lancefield group B
Acid-stable polysaccharide in the cell wall
Capsular polysaccharide serotypes
o Can be detected by precipitin test
Sialic acid composition of the terminal position (repeating unit)
Virulence Factors
Capsule important virulence factor
o Prevents phagocytosis
o Ineffective after opsonisation
Sialic acid critical virulence determinant
o Most significant component of the capsule
o Loss of capsular sialic acid = loss of virulence
Clinical Infections
Cause of mastitis in cattle
Invasive disease in newborn
Neonatal Sepsis
o Early-onset infection (less than 7 days old)
Vertical transmission from the mother
Pneumonia or meningitis with bacteremia
Presence of group B streptococci in the vagina of the mother
o Late-onset infection (atleast 7 days old)
Meningitis
Uncommonly associated with obstetric complications
2 Types of infections in adults
o Young previously healthy women who became ill after childbirth or abortion
Endometritis and wound infections
o Elderly serious underlying disease or immunodeficiency
Treatment: Ampicillin + Aminoglycoside

Laboratory Diagnosis
Grow on SBA: grayish white mucoid colonies surrounded by a small zone of -hemolysis
Gram (+) cocci, short chains (clinical specimens) or longer chains (culture)
Useful tests: Hippurate hydrolysis or CAMP test
StrepB Carrot Broth: (+) result is orange or red pigment

Groups C and G Streptococci

Classified with pyogenic streptococci
S. dysgalactiae subsp. equisimilis large colony forming hemolytic
S. anginosus small colony forming hemolytic
S. equi subsp. zooepidemicus animal pathogen
Uncommon human pathogens

Streptococcus pneumoniae

Antigenic Structure
Also known as pneumococcus
Contains antigen C substance (similar to C carbohydrate)
C-reactive protein (-globlulin in human serum) + C substance = precipitate
o A chemical reaction NOT an antigen-antibody combination
o CRP increases during inflammation and infection
Virulence Factors
Capsular polysaccharide
Loss of ability to produce capsule = non-pathogenic
Opsonisation of the capsule = avirulent
Clinical Infections
Causes pneumonia, sinusitis, otitis media, bacteremia and meningitis

Under the age of 3 years old or elderly with underlying disease

Pneumonia is not usually a primary infection but rather a result of disturbance of the
normal defense barriers
o Alcoholism, anesthesia, malnutrition and viral infections lobar pneumonia
o Sudden onset with chills, dyspnea and cough
o May be complicated by a pleural effusion; usually sterile (empyema)
Vaccinations: PCV7 (children) and PS23 (adults)
Treatment: erythromycin or chloramphenicol

Laboratory Diagnosis
Gram stain: gram (+) in pairs; ends of the cells are slightly pointed (oval/lancet shape)
The capsule can be demonstrated using a capsule stain
On SBA
o Young cultures: round, glistening, wet, mucoid, dome-shaped appearance
o Older cultures: collapse of colonys center, appearance of a coin w/ a raised rim
o To differentiate it from Viridans optochin susceptibility and bile solubility
Viridans Streptococci
hemolytic
Normal microbiota of the upper respiratory tract, female genital tract and GIT
Viridans = green
Fastidious, some strains requiring CO2 for growth
5 groups
o S. mitis
o S. bovis
o S. mutans
o S. anginosus
o S. salivarius
Group D streptococci was subdivided into 2 groups bile esculin (+)
o Enterococcal Enterococcus; found in the intestinal tract
o Nonenterococcal would not grow in a nutrient broth w/ 6.5% NaCl
Clinical Infections
Oropharygeal commensals opportunistic pathogens
Oral infections such as gingivitis and dental caries (S. mutans)
Bacterimic patients with the tendency to form abscesses (S. anginosus)
Bacterial endocarditis in native valves (S. mitis)
Blood cultures of patients with bacteremia, septicaemia, and endocarditis (S. bovis)
Virulence Factors
Polysaccharide capsule and cytolysin in some members of the anginosus group
Extracellular dextran and cell surface-associated proteins
Some possess similarity to Group A
Laboratory Diagnosis
All members are PYR (-) and LAP (+)
Colonies are small, surrounded by a zone of -hemolysis; some are or nonhemolytic

S. anginosus

o minute colony types

o When growing in pure culture or high concentration sweet odor of
honeysuckle or butterscotch

Enterococcus

Natural inhabitants of the intestinal tracts

Lancefield group D
Sometimes produce pseudocatalase production weak bubbling in catalase test

Virulence Factors
Ability to grow under extreme conditions
Resistant to multiple antimicrobial agents
Cytolysin quorum-sensing mechanism

Clinical Infections
Frequent causes of nosocomial infections
UTIs associated with urinary catheterization or other urologic manipulations
Prolonged hospitalization risk factor for acquiring bacteremia
Endocarditis seen mainly in elderly patients
Laboratory Diagnosis
Culture: Trypticase Soy or Brain-heart infusion agar supplemented with 5% sheep
blood
Identified based on their ability to:
o Produce acid in carbohydrate broth
o Hydrolyze arginine
o Tolerate 0.04% tellurite
o Utilize pyruvate
o Produce acid from methy--d-glucopyranoside (MGP)
o Growth around 100 g efrotomycin acid disk
o Exhibit motility
E. faecalis
o Easily identified by its ability to grow in the presence of tellurite

Streptococcus-like Organisms
1. Abiotrophia and Granulicatella

Nutritionally variant streptococci

Viridians streptococci
Cysteine or pyridoxal (Vit B6)
Thiol or pyridoxal requiring
Grow as satellite colonies around other bacteria
Require sulfydryl compounds for growth
Bile esculin (-), no growth in 6.5% NaCl
PYR positive
Gram (+) cocci, short chains
Significant cause of bacteremia, endocarditis and otitis media
Increased tolerance to antimicrobial agents
o Surgery to achieve cure

2. Aerococcus

Common airborne organism

Widespread, opportunistic pathogen associated with bacteremia, endocarditis
and UTI
Occur as tetrads or clusters in broth media
Grow in the presence of 6.5% NaCl
Can be easily confused with Enterococci
A. viridians (bile esculin and PYR +)
A. urinae (bile esculin and PYR -)

3. Gemella

Similar in colony morphology and habitat to the viridians streptococci

-hemolysis or nonhemolytic
Easily decolorize on gram staining Gram (-) cocci in pairs, tetrads, clusters or
short chains

4. Lactococcus

Gram (+) cocci in singly, pairs or chains

Previously classified as group N streptococci
UTI and endocarditis
Production of acid from carbohydrates
Do not react with the genetic probe in the AccuProbe Enterococcus Culture
Confirmation Test

5. Leuconostoc

Catalase (-), Gram (+) with irregular coccoid morphology

Intrinsically resistant to Vancomycin
Plant surfaces, vegetables and milk products
Meningitis, bacteremia, UTI and pulmonary infection
Absence of PYR and LAP activities, hydrolysis of esculin in the presence of bile,
growth in 6.5% NaCl and production of gas from glucose

6. Pediococcus

Facultatively anaerobic, Gram (+)

Intrinsically resistant to Vancomycin
Patients w/ GIT abnormalities or have previously undergone abdominal surgery
Bile esculin (+), presence of LAP activity and absence of PYR activity

Laboratory Diagnosis
A. Hemolytic Patterns (Refer to Hemolysis table)
B. Physiologic Characteristics
1. Pyogenic
Pus producing
hemolytic
Lancefield grouping
2. Viridans
or nonhemolytic
Opportunistic pathogens
Not part of Lancefield group
Upper respiratory tract
3. Lactic Acid
Nonhemolytic
Dairy products
Lancefield group N

4. Enterococci
Flora of the intestine
Resistant to Vancomycin
C. Lancefield Classification Scheme (Refer to table in Cell Wall Structure)
Based on Extraction of C-carbohydrate
o Placing of organism in dilute HCl
o Heating for 10 minutes
D. Biochemical Identification

a. Bacitracin Susceptibility
Identify Group A Streptococci in throat cultures
Inoculate SBA containing SXT (Sulfamethaxozole and Trimethoprim)
and Bacitracin disk is placed directly onto the agar
o GROUP A: susceptible to bacitracin; resistant to SXT
o GROUP B: resistant to bacitracin and SXT
b. CAMP Test
Identify Group B Streptococci
Christie, Atkins, and Munch-Petersen

Streaked perpendicularly to a -hemolytic S. aureus

o Result: arrowhead zone of hemolysis

c. Hippurate Hydrolysis
Group B vs -hemolytic streptococci
S. agalactiae (hippuricase or hippurate hydrolase) hydrolyzes Na
hippurate to Na benzoate and glycine ninhydrin reacting with amino group (purple color)
d. PYR Hydrolysis
Pyrrolidonyl--naphthylamide hydrolysis
Identification of -hemolytic group A Streptococci and nonhemolytic
Group D
Takes advantage of the fact that S. pyogenes and Enterococcus spp. are
able to hydrolyze the substrate PYR
More specific for S. pyogenes than bacitracin
e. Leucin Aminopeptidase (LAP)
Hydrolyzes peptide bonds adjacent to a free amino group
Reacts most quickly with leucine (leucine aminopeptidase)
Leucine- -naphthylamide is hydrolyzed to -naphthylamine
Addition of para-dimethylaminocinnamaldehyde reagent (DMACA)
red color develops
Differentiating Aerococcus and Leuconostoc (LAP -) from other gram (+)
cocci
f. Voges-Proskauer Test
Distinguishing small colony-forming -hemolytic anginosus group from
large colony forming pyogenic strains of A or C antigens
Detects acetoin production from glucose
(+) reaction: formation of red or pink color
g. -D-Glucuronidase (BGUR) Test
detects the action of BGUR enzyme found in isolates of large colonyforming -hemolytic groups C and G
h. Bile Esculin and Salt Tolerance
Identification for the nonhemolytic, catalase (-), gram (+) cocci
Bile Esculin
o Detecting growth of bacteria in the presence of 40% bile
o Ability to hydrolyze esculin
Salt Tolerance
o Enterococcus and Aerococcus growth in 6.5% NaCl broth
o Group D no growth

i.

Optochin Susceptibility
Filter paper disk containing optochin (ethylhydrocuprein hydrochloride)
is added to the surface of SBA plate
o Zone of inhibition: > than 14-16mm susceptible (S. pneumoniae)

j.

Bile Solubility
Takes advantage of the very active S. Pneumoniae autocatalytic
enzyme amidase
Bile salt or detergent cell wall lyses during cell division (clear)
Suspension of S. pneumoniae in a solution of sodium deoxycholate lyses
solution becomes clear

k. Antigen Testing
Capsular polysaccharide
Specific antisera for S. pneumoniae tagged with Latex particles
o Agglutination presence of corresponding capsular antigen
l.

Growth in 6.5% NaCl

Group D enterococci (salt tolerant)
Non-enterococci (intolerant)
6.5% salt broth + Bromcresol purple purple or yellow or appearance
of obvious growth

Noncultural Identification
Immunoassays
o Identification of streptococci particularly Group A
Detection of the group-specific antigen from either isolated colonies or
direct specimen such as throat swab
Capillary precipitation or slide agglutination test

Nucleic Acid Probes

o Rapid results and increased specificity
o More costly
o The LightCycler real-time polymerase chain reaction (PCR)
Detects the cfb gene that encodes the CAMP-factor protein of Group B
streptococci and the ptsI (phosphotransferase) gene of Group A
streptococci

Alpha Hemolytic Streptococci

GROUP

Optochin

Bile Esculin

6.5% NaCl

PYRase

D Enterococcus
D Nonenterococcus
S. Pneumoniae
S. Viridans

R
R
S
R

+
+
-

+
V

+
-

Beta Hemolytic Streptococci

GROUP

CAMP

Bile Esculin

6.5% NaCl

PYRase

A
B
C, F, G
D Enterococcus
D Nonenterococcus

S
R
R
R
R

R
R
S
R
R

+
-

+
+

+
-

+
+
-

Nonhemolytic Streptococci
GROUP

Bile Esculin

6.5% NaCl

PYRase

D Enterococcus
D Nonenterococcus

+
+

+
-

+
-

ENTEROBACTERIACEAE
General Characteristics
Often referred to as enterics
Consists of a large number of diverse organisms
Encountered in Clinical Laboratory
Associated with personal hygiene (fecal-oral route), poor sanitation and colonization
(skin and respiratory tract)
Glucose fermenters
Cytochrome oxidase negative
Reduce nitrates to nitrites
o Erwinia, Pantoea agglomerans
Mostly motile Pili (fimbriae)
Microscopic and Colony Morphology
Gram (-), non-sporeforming, facultative anaerobic bacilli
Gram stained smears: coccobacilli or as straight rods
Sheep blood agar or Chocolate agar: little value for identification
Colonies: large, moist, gray indistinguishable
o Many isolates of E. coli are -hemolytic
Blood agar: Dull gray; variable hemolysis
MAC, Hektoen Enteric (HE) and XLD agar presumptive identification
o Contain one or more carbohydrates (lactose and sucrose)
o Show the ability of the species to ferment specific carbohydrates
Fermentation indicated by color change in the medium
Drop in pH indicated by a pH indicator
Nonfermenting sp. - lack of color change and retention of medium color
H2S producing sp. distinguished when placed in HE or XLD agar
Contain sodium thiosulfate and ferric ammonium citrate
Blackening of H2S producing colonies
Lactose Fermentation
Rapid Fermenting
o Escherichia
Late Fermenting
o Serratia
o Hafnia
Non-Fermenting
o Shigella
o Salmonella
o Proteus

o Enterobacter

o Klebsiella

o Pantoea
o Citrobacter
o Providencia
o Morganella
o Edwardsiella

o Yersinia

Virulence Factors
Ability to adhere, colonize, produce toxins, and invade tissue
Some species harbor plasmids that can provide antimicrobial resistance genes

Differential and Selective Media

Medium

Inhibitor
Crystal violet
Bile salt
Eosin Y
Methylene blue

Carbohydrate

Indicator

Lactose

Neutral red

Lactose

Eosin Y
Methylene blue

Desoxycholate

Bile salt

Lactose

Neutral red

XLD

Bile salt

Lactose
Xylose
Sucrose

Phenol red

Desoxycholate
citrate

Bile salt

Lactose

Neutral red

Bile salt

Lactose
Sucrose
Salicin

Bromothymol
blue

(+) red
(-) colorless
(+) orange
(-) green w/ or w/o black

Neutral red

(-) black w/ metallic

sheen
(-) colorless w/ or
w/o black center

MAC
EMB

Hektoen Enteric
Bismuth Sulfite
Salmonella-Shigella

Brilliant green
Bismuth sulfite
Bile salt
Sodium citrate
Brilliant green

Glucose
Lactose

Color of the Colony

(+) pink red
(-) colorless
(+) dark purple w/ sheen
(-) transluscent
(+) red
(-) colorless
(+) red
(-) colorless

Serologic Characteristics
O antigen or somatic antigen
o Heat-stable antigen
o Located on the cell wall
o Lipopolysaccharide (LPS)
H antigen or flagellar antigen
o Heat-labile antigen found on the surface of flagella
o Structures responsible for motility
o Protein
K antigen or capsular antigen
o Heat-labile
o Found in capsular polysaccharide
o Covers the O antigen
o Examples: K1 antigen of E. coli and Vi antigen of Salmonella enterica
Clinical Significance
Ubiquitous
Normal flora of the GI tract
Often commensals causing no harm
2 categories:

o Opportunistic pathogens
Usual intestinal microbiota
May produce serious extraintestinal, opportunistic infections
Example: E. coli
UTIs, gastroenteritis, septicemia, wound infections, meningitis
Poultry, livestock, fish, vegetable crops
o Primary pathogens
True pathogens
Not present as commensal biota in the GI tract
TSI or Triple Sugar Iron Agar
Yellow (acid) deep
o Glucose fermentation
Yellow (acid) slant
o Lactose and/or sucrose fermentation
Blackening in the butt
o H2S production
Crack in the medium
o Gas production
TSI Reactions
Reaction
A/A

K/A H2S+

K/A H2S-

K/K H2S-

A/A H2S+

Carbohydrate Fermented
Glucose w/ acid and gas
Lactose and sucrose w/ acid
and gas
Glucose w/ acid and gas
Lactose or sucrose not
fermented
Glucose w/ acid; no gas
Lactose or sucrose not
fermented
Glucose not fermented
Lactose or sucrose not
fermented
Glucose fermented w/ gas
Lactose or sucrose not
fermented

LIA or Lysine Iron Agar

Lysine decarboxylase positive
o Purple/purple
Lysine decarboxylase negative
o Purple/yellow

Typical organisms

Escherichia
Klebsiella
Enterobacter
Salmonella, Proteus
Citrobacter, Arizonae
Edwardsiella
Shigella, Serratia
Salmonella, Proteus
Providencia
Pseudomonas
Alcaligenes
Citrobacter freundii

Deaminase positive
o Red/yellow
H2S positive
o Blackening

IMViC Tests
1. Indol production test
Organism is grown in Tryptone broth
Enzyme: Tryptophanase
o Tryptophan Indole molecule, pyruvate and CO2
o Indole is extracted by means of xylene
To test for Indole production: Kovacs or Ehrlichs reagent
o Contains para-aminobenzaldehyde
Results: (+) red ring; (-) yellow or brown ring
2. Methyl red test
MRVP broth
Detects production of acids formed during metabolism
o Mixed acid fermentation pathway
o Substrate: pyruvate
o pH indicator: Methyl red (+)
o If solution remains yellow (-) butanediol fermentation
3. Voges-Proskauer test
MRVP broth
Uses alpha-naphtol and potassium hydroxide to test for presence of acetoin
2,3-butanediol fermentation pathway
Result: (+) pinkish-red

4. Citrate utilization test

Uses Simmon Citrate agar (contains citrate and ammonium ions)

Determine the ability of a microorganism to use citrate as its sole carbon source
Degradation of citrate
Indicator: Bromothymol blue
o Green to blue
o Result: (+) blue; (-) green

Urease Test
Urea broth or Urea Christensen agar
Indicator: Phenol red
Results: (+) red color; (-) salmon color or yellow color
Urea is hydrolyzed by urease = CO2 + ammonia
Phenylalanine Test
Used to determine if the microbe produces the enzyme phenylalanine deaminase
Determined by the deamination of phenylalanine to phenylpyruvic acid
Medium used is phenylalanine agar
10% ferric chloride is added Result: (+) green color in the syneresis fluid
ONPG or Ortho-nitrophenyl-beta-D-galactopyranoside
Slow or late lactose fermenter
Salmonella arizonae only one that is ONPG positive; Salmonella serotype
Citrobacter positive
Lactose Fermenters

Nonlactose Fermenters

No Spreading or Swarming

Enterotube
A sample of multi-test system

ID Value

70742
70743
70762
70763

Organism

Atypical Test Results

K. pneumoniae

VP (-); Citrate (-)

VP (-)
Citrate (-)
none

Escherichia coli

Serotypes: 06, 08, 015, 025, 063, 078, 0148, 0159

Human GI tract
o Commonly isolated from the colon
Wide range of clinical syndromes, UTIs, CNS infections and diarrheal disease
Primary marker of fecal contamination in water purification
Motile and possess adhesive fimbriae, sex pili and antigens
o Remarkable cross-reactivity with O antigens
o Closely related to Shigella (DNA homology)
o O and H antigens: identification of strains associated with serious enteric disease
o K antigen: masks O antigen during bacterial agglutination by specific antiserum
Properties:
o Fermentation of glucose, lactose, trehalose and xylose
o Indole production from tryptophan
o Glucose fermentation by the mixed acid pathway: MR (+) and VP (-)
o Does not produce H2S, DNase, urease or phenylalanine deaminase
o Cannot use citrate as a sole carbon source

Uropathogenic Escherichia coli

Most common cause of UTIs in humans
o E. coli strains that originate in lasrge intestine as resident biota
Produce factors that allow them to attach urinary epithelial mucosa
o Virulence Factor:
Production of pili which allow strains to adhere epithelial cells and not
be washed out with urine flow
Cytolysins kill immune effector cells and inhibit the phagocytosis and
chemotaxis of certain WBC
Aerobactin allows the bacterial cell to chelate iron; free iron is
generally unavailable within the host for use by bacteria
E. coli strains that cause acute pyelonephritis
o Dominant resident in the colon
o Resistant to the antibacterial activity of human serum
Gastrointestinal Pathogens
Enterovirulent E. coli or diarrheagenic E. coli
o Enterotoxigenic Escherichia coli or ETEC
Diarrhea of infants and adults in tropical and subtropical climates
Travelers diarrhea, Montezumas Revenge or Turista
Spread via consumption of food or water contamination
Major contributing factors: poor hygiene, reduced sources of potable
water and inadequate sanitation
Stomach acidity inhibiting colonization (small intestine) and initiation
of disease
Heat-labile toxin (LT)
Two fragments:
- A (active)
Activates cellular adenylate cylase
ATP cAMP
Hypersecretion of electrolytes and fluid watery
diarrhea
- B (moiety or binding; confers specificity)
Providing entry for the A portion
Heat-stable toxin (ST)
- Stimulates guanylate cylase increased production of cGMP
Severe epidemic diarrhea = cholera, fever, headache, chills
o Enteroinvasive Escherichia coli or EIEC
Serotypes: 028, 0112, 0115, 0124, 0136, 0143, 0144, 0147, 0152
Produce dysentery with direct penetration, invasion and destruction of
the intestinal mucosa
Disease: Shigellosis
Direct transmission: fecal-oral route
Fever, severe abdominal cramps, malaise and bloody stool with RBCs,
neutrophil and mucus
Mostly nonmotile (Late LF or NLF)

Cross reaction between shigellae and EIEC O antigens

Definitive identification: enteroinvasiveness
Sereny Test - previously used to determine virulence of shigellae & EIEC
Monolayer cell cultures with human epithelial-2 cells (Hep-2) - detect
invasiveness

o Enteropathogenic Escherichia coli or EPEC

Infantile diarrhea
Low-grade fever, malaise, vomiting, and diarrhea
Stool = large amounts of mucus but no blood
Noninvasive, no toxin production, intimin
To identify: serologic typing with pooled antisera
o Enterohemorrhagic Escherichia coli or EHEC
0157:H7 strain of E. coli
Verotoxic E. coli (VTEC)
Bloody diarrhea (verotoxin) hemorrhagic colitis, Hemorrhagic
diarrhea, and hemolytic uremic syndrome (HUS)
HUS = low platelet count, haemolytic anemia, and kidney failure
Potentially fatal
Produces 2 cytotoxins
Verotoxin I
- Phage-encoded cytotoxin identical to Shiga toxin (Stx)
- Produces damage to Vero cells
- Reacts with and is neutralized by the antibody against Stx
Verotoxin II
- Biologically similar but immunologically different from Stx
and verotoxin I
- Also called Shiga toxigenic E. coli
May be identified by:
Stool culture on highly differential medium w/ subsequent
serotyping
Detecting the verotoxin in stool filtrates
Demonstration of a fourfold or greater increase in verotoxinneutralizing antibody titer
SMAC MAC agar containing sorbitol
Stool culture for E. coli 0157:H7 (appears colorless)
o Enteroadherent Escherichia coli
Diffusely adherent E. coli or DAEC
Associated with both UTIs and diarrheal disease
Cystisis in children and acute pyelonephritis in pregnant women
Chronic or recurring UTIs
Enteroaggregative (EAEC)
Causing diarrhea by adhering to the surface of the intestinal
mucosa (Hep2 cells)
Packed in an aggregative stacked brick pattern
Watery diarrhea, vomiting, dehydration and abdominal pain

Extraintestinal Infections
One of the most common causes of septicemia and meningitis among neonates
o Infection usually acquired in the infection of the birth canal (mothers vagina
usually heavily colonized)
Virulence factor: Capsular antigen K1
Significant isolate in blood cultures from adult
E. coli bacteremia in adults: urogenital tract infection or from a GI source
Other Escherichia Species
Escherichia hermannii
o E. coli atypical or enteric group II
o Yellow-pigmented organism
o Isolated from CSF, wounds and blood
Escherichia vulneris
o Isolated from humans with infected wounds
o Yellow-pigmented colonies
Escherichia albertii
o Associated with diarrheal disease in children
Tribe Klebsiellae
Genus Klebsiella, Enterobacter, Serratia, Pantoea, Cronobacter, and Hafnia
Usually found in the intestinal tract or free-living
Associated with a wide variety of opportunistic and nosocomial infections
Characteristics:
o Most grow on Simmons citrate and in potassium cyanide broth
o None produce H2S
o A few hydrolyze urea slowly
o All give a (-) reaction with MR and a (+) reaction on VP test
o Few exceptions, indole is not produced from tryptophan
o Motility is variable

Klebsiella

Usually found in the GI tract

Short and nonmotile
Strings out when touched by the needle

K. oxytoca

K. pneumoniae

o Most commonly isolated

o Polysaccahride capsule (Encapsulated)
Protection against phagocytosis and antimicrobial absorption
Responsible for the moist, large, mucoid colonies
o Frequent cause of Lower RT infections among hospitalized patiend and
immunocompromised hosts
o Bacterial pneumonias
o Identical to K. pneumoniae
o Blood and stool culture

o Indole positive and ornithine positive isolates

K. ozaenae

K. rhinoscleromatis

o Atrophy of the nasal mucous membranes with fetid odor

o Blood and stool culture

o Rhinoscleroma: infection of the nasal cavity that manifests as an intense swelling

and malformation of the entire face and neck
o Destructive granuloma of the nose and pharynx
K. ornithinolytica (indole and ornithine decarboxylase positive) and K. planticola
o Have been isolated from urine, RT and blood

Enterobacter, Cronobacter, and Pantoea

Motile
ODC (Ornithine Decarboxylase broth) positive
Grow on Simmons citrate medium and in potassium cyanide broth
MR test (-) and VP test (+)
Lysine decaboxylase (+) except E. gergoviae or E. cloacae

E. gergoviae

E. aerogenes

E. sakazakii

E. cloacae

o Predominant isolate
o UTI and wound infection
o Resembles E. aerogenes
o Strong urease activity
o Endocarditis and meningitis

o Resembles E. cloacae
o Yellow pigmented
o Neonatal sepsis
E. taylorae (formerly name)
o E. cancerogenus
o Osteomyelitis following traumatic wounds
o Lactose negative
o ONPG positive

Pantoea agglomerans

o Contaminated IV fluids
o Yellow pigment
o Negative to ADH, LDH, ODC

Serratia

Opportunistic pathogens
Ferment lactose slowly and ONPG (+)
o Except S. fonticola
Deoxyribonuclease, lipase, gelatinase
Ability to produce extracellular DNase
Resistance to a wide range of antimicrobials

S. marcescens, S. rubidaea, and S. plymuthica

o Pink to red pigment, prodigiosin especially when incubated at room temp.

o S. marcescens
Clinically significant
Pneumonia and septicemia
S. liquefaciens: ferment arabinose
S. odorifera: Rancid, potato-like odor; (+) for sucrose, raffinose and ornithine

Decarboxylase Reactions

K. pneumoniae
K. oxytoca
E. aerogenes
E. cloacae
P. agglomerans

LDC
+
+
+
-

ODC
+
+
-

ADH
+
-

Hafnia

Isolated from a number of anatomic sites in humans

Not known to cause gastroenteritis but occasionally isolated from stool cultures

H. alvei biotype 1

H. alvei

o Negative to lactose, sucrose, sorbitol, citrate, DNase, lipase

o Grows in the beer wort of breweris; not isolated clinically

Tribe Proteeae
Genera: Proteus, Providencia and Morganella
Normal intestinal microbiota
Opportunistic pathogens
Distinguished by its ability to deaminate the amino acid phenylalanine (Deaminase +)
KCN growth (+)
MR (+), LDC (-), ADH (-)
Motile
Does not ferment lactose

Proteus

Ascend in urinary tract infections in lower and upper UT

Infect proximal kidney tubules and can cause acute glomerulonephritis
UTIs and wound infections
Soil, water and focally contaminated materials
Rapid urease activity
P. mirabilis and P. vulgaris
o Swarming motility on BA

Result of a tightly regulated cycle of differentiation from standard

vegetative cells (swimmers) to hyperflagellated, elongated, polyploidy
cells (swarmers) coordinated surface movement
Distinct odor: burnt chocolate
o H2S (+) and hydrolyze urea
o Differentiated by:
P. mirabilis: (-) indole, (+) ornithine
P. vulgaris: (+) indole, (-) ornithine, ferment sucrose acid/acid reaction
in TSI agar

P. penneri

P. myxofasciens

o Isolated from patients with diarrhea

o Isolated only from gypsy moths
o Characterized by the large amount of slime it produces

Differentiation of the Tribe Proteeae

TSI
H2S
Gas
MR
VP
Indole
Citrate
Deaminase
Urease
Motility
LDC
ADH
ODC

P. vulgaris
K/AG
+
+
+
+
+
+
S/+
-

P. mirabilis
K/AG
+
+
+
V
V
+
+
S/+
+

M. morganii
K/AG
+
+
+
+
+
+
+

P. rettgeri
K/A
+
+
+
+
+
+
-

P. stuartii
K/A
+
+
+
+
+
-

Differentiation of Proteus
Reaction
Indole
ODC
Maltose
D-xylose
Salicin
Chloramphenicol

mirabilis
+
+
S

vugaris
+
+
+
+
R

penneri
+
+
R

myxofaciens
+
S

Morganella
M. morganii: documented cause of UTI but has not been isolated
Providencia
P. rettgeri: UTI and occasional nosocomial outbreaks
P. stuartii: nosocomial outbreaks in burn units; isolated form urine cultures
Edwardsiella
E. tarda

Human pathogen
Negative for urea
Source of GI infection, wound infections and abscesses
Trauma or accidents (aquatic environment)
Biochemically similar to E. coli
Positive for LDC, ODC, H2S (abundant in TSI agar) and indole
Does not grow on Simmons citrate
E. hoshinae: snakes, birds and water
E. ictaluri: enteric septicaemia in fish
o
o
o
o
o

Erwinia and Pectobacterium

Plant pathogens not significant in human infections

Citrobacter

Resembles salmonella
o Do not decarboxylate lysine
o ONPG (+) and LDC (-)
Resembles E. coli
o Colonies produced on MAC Hydrolyze urea slowly & ferment lactose
o Produce H2S
All species grow in Simmons citrate test and (+) in MR test
Late lactose fermenters
Associated with nosocomial infections and UTI

C. freundii

Can be isolated in diarrheal stool cultures

A known extraintestinal pathogen
Associated with endocarditis in IV drug abusers
Motile
(+): gas production, MR, citrate, H2S and KCN growth
(-): VP and deaminase
Urease and ADH variable
C. diversus or koseri
o Nursery outbreaks of neonatal meningitis and brain abscesses
o Indole (+)
o
o
o
o
o
o
o

H2S

C. freundii
+

C. diversus
-

Indole
ODC
KCN (Growth)

V
+

+
+
-

Salmonella

Very complex genus

Kauffman-White Classification (2200+ serotypes)
Colonies:metallic colonies with black ring (BSA and BGA)
Biochemical Features:
o Do not ferment lactose
o (+): H2S
o (-): indole, VP test, phenylalanine deaminase and urease
o Do not grow in medium containing potassium cyanide
Virulence Factors
o Fimbriated strains appear more virulent than nonfimbriated strains
o Ability to traverse intestinal mucosa
o Enterotoxin
Antigenic Structures
o Somatic O antigens (Group A, B, C, D)
o Flagellar H antigen (1, 2, 3, 4) occur in phases
Phase 1: occur only in a small number of serotypes and determine the
immunologic identity of the particular serotype; antigens agglutinate
only w/ homologous antisera
The specific phase
Phase 2: occur among several strains, shared by numerous serotypes,
react with heterologous antisera
The nonspecific phase
o Vi antigen
Surface polysaccharide capsular antigen
Preventing phagocytosis
Often block O antigen during serologic typing
Can be removed by heating
Clinical Infections
o Pathogenic
o Infection from ingestion of contaminated foods and contaminated hands
o Salmonellosis
o Acute gastroenteritis or food poisoning vomiting and diarrhea
Symptoms appear 8-36 hours after ingestion
Nausea, vomiting, fever and chills
Treatment: chloramphenicol, ampicillin and trimethoprimsulfamethoxazole
o Typhoid fever (severe enteric fever)
Prolonged fever (1 week)
Nontyphoidal bacteremia
Dissemination to multiple organs
Develops 9-14 days after ingestion

Symptoms depend on the no. of organisms ingested: the larger the

inoculums, the shorter the incubation period
1st week
Fever, vomiting and headache
Constipation
2nd to 3rd week
Bacteremia
Rose spots
Invades gallbladder and Peyers patches of the bowel
Lymphoid tissue and RES (intracellularly within the monocytes)
Blood (1st and 2nd week)
Urine (3rd and 4th week)
Stool (indefinite)

Clinical Diseases induced by Salmonella

Incubation period
Onset
Fever
Duration of
disease
GI symptoms
Blood cultures
Stool cultures
o
o
o
o

Enteric Fevers
7-20 days
Insidious
Gradual then high
plateau with "typhoidal"
state
Several weeks
Often early constipation;
later bloody diarrhea
+ in 1st and 2nd week
+ from 2nd on; - earlier
in disease

Septicemia
variable
abrupt

Gastroenteritis
8-48 hours
abrupt

Rapid rise, then spiking

"septic" temperature

Usually low

variable

+ during high fever

2-55 days
nausea, vomiting,
diarrhea at onset
negative

infrequently positive

positive soon after onset

often none

S. typhi: enteric fever; typhoid and paratyphoid

S. choleraesuis: septicemia
S. typhimurium and S. enteritidis: gastroenteritis

Widal Test
O antigen: active infection
H antigen: past immunization or infection
Vi antigen: carriers
o Limulus Test (Endotoxin) amebocytes of horse crab (visible gel)
o Ideal Loop Test (Enterotoxin) ballooning of the intestinal tract or ileal loop
(accumulation of fluid)

Shigella

Resembles E. coli
Nonmotile and lactose negative
Do not produce gas from glucose except S. flexneri

Do not hydrolyze urea

Do not produce H2S
Do not decarboxylate lysine
Fragile organisms
Shigellosis: bacillary dysentery
o Bloody, mucoid diarrheic stool with pus cells and fishy odor

S. sonnei

S. dysenteriae

o Ability to decarboxylate ornithine

o Slowly ferments lactose (delayed) pink colonies on MAC
o (+): ONPG and ODC

o Most prevalent in the US

o Beurotoxin and enterotoxin
o Non-mannitol fermenter
Other species: S. flexneri and S. boydii

Mannitol
fermentation
ONPG
Ornithine
decarboxylase
Serogroup

dysenteriae

flexneri

boydii

sonnei

Yersinia

Small coccobacilli
Pinpoint colonies on MAC
TSI = yellow/orange (weak acid in the slant, no change in the butt)
Nonmotile at 37 except Y. Pestis (motile at RT)

Yersinia enterocolitica

Yersinia pestis

o Gram (-), short, plump bacillus

o Methylene blue or Wayson stain
Bipolar staining (intense staining)
Safety-pin appearance
o Plague (black death)
o Disease primarily of rodents, squirrels and rabbit
Transmitted to humans by bites of fleas, hadling of infected animal
Stalactite patterns
o Forms: Bubonic or glandular, septicemic and pneumonic form
o Symptoms may appear 2-5 days after infection
o High fever w/ painful regional lymph (buboes)
o Enterocolitis
o Ingestion of contaminated water, pork, beef or milk products
o Fecal-oral transmission

o Ferments mannitol and appears as red, bulls eye colonies with colorless halo
(CIN medium)
o ODC positive ferments both sucrose and sorbitol

Yersinia pseudotuberculosis

o Pathogen primarily of rodents (esp. Guinea pigs), wild animals and game fowl
Caseous swellings: pseudotubercles
o When ingested: spread to the mesenteric lymph nodes generalized infection
o Appears as typical-lookingplague bacillus
o Lymphadenitis in children

Differentiation of Genus Yersinia

Test
Indole
Methyl red
Voges-Proskauer
25C
37C
Motility
25C
37C
-Galactosidase
Phenylalanine
deaminase
Ornithine
decarboxylase

pestis
+

enterocolitica
d
+

pseudotuberculosis
+

d
-

+
+

+
+

Other Genera of the Family Enterobacteriaceae

Budivicia
Budivicia aquatic

Usually found in water; occasionally occur in clinical speciemens

Buttiauxella
B. agrestis and B. noackiae found in human specimens
Similar to both Citrobacter and Kluyvera except the DNA hybridization
Cedecea

Recovered from sputum, blood and wounds

C. davisae most commonly isolated

Ewingella
E. americana only specie

Isolates from human blood cultures or respiratory specimens

Kluyvera

Species: ascorbata, cryocrescens and Georgiana

Found in respiratory, urine and blood cultures
Produce a blue-violet pigment
Resembles E. coli colonies growing on MAC agar
To differentiate from one another
Cephalothin and carbenicillin disk susceptibility tests (separates the first 2 species)
o ascorbata: small zones, does not ferment glucose
o cryocrescens: large zones, ferments glucose at 5C
o georgiana: (-) for gas production during fermentation of glucose

Leclercia
L. adecarboxylata yellow pigment in initial isolation
Similar IMViC reactions to E. coli

(-): LDC, ODC and ADH

Leminorella

Species: L. gromontii and L. richardii

(+): H2S and weak reactions with Salmonella sera
Inactive and clinical significance is unknown

Moellerella
M. wisconsensis

(+): citrate, MR, lactose and sucrose

(-): LDC, ODC, ADH and indole

Obesumbacterium

Closely related to Escherichia blattae

Fastidious and slow-growing organisms

Photorhabdus
P. luminescens

o Subspecies: luminescens, akhurstii and laumondii

P. asymbiotica and P. temperate
Natural habitat: lumen of entomopathogenic nematodes
Pink/red/orange/yellow or green pigmented colonies on NA
(-): nitrate reduction

Pragia
Pragia fontium

(+) H2S and MR, Shigella-like odor (NA) and a pig sty-like odor (Endo agar) w/ a
sloping surface of agar
Utilize citrate and oxidation of gluconate

Rahnella
Rahnella aquatilis water bacteria that are psychotolerant

No single characteristic that distinguishes them from other Enterobacteriaceae

Tatumella
Tatumella ptyseos

Slow-growing, tiny colonies producing and nonreactive

Trabulsiella
Trabulsiella guamensis

Biochemically similar to Salmonella

Isolated from vacuum-cleaner contents on the island of Guam

Xenorhabdus
X. nematophilus isolated form nematodes
Yokenella
Yokenella regensburgei

(-): VP test

NEISSERIA SPECIES AND MORAXELLA CATARRHALIS

General Characteristics
Gram (-), Catalase (+), aerobic, nonmotile, nonspore-forming diplococci
o N. elongata catalase (-), rod shaped
o N. weaver catalase (+), rod shaped
Oxidase Test: cytochrome oxidase oxidase (+)
Increased CO2 for growth (capnophilic)
o Optimal growth in humid atmosphere
Anaerobic if alternate electron acceptors are available
Habitat: mucous membranes of the respiratory and urogenital tracts
Opportunistic pathogens except for the 2 primary human pathogens:
o N. gonorrhoeae and N. meningitidis
Require iron for growth
Compete with their human host by binding transferrin
Primary reason that they are strict human pathogens
Fastidious, requiring enriched media for optimal recovery

Neisseria gonorrhoeae

Humans natural host

Always pathogenic
Not part of the normal biota
Agent of gonorrhea

Virulence Factors
Receptors of human transferrin
Pili (fimbriae)
o Initial attachment
o Inhibit phagocytosis by neutrophils
o Aid in the exchange of genetic materials between cells
o 5 Colony Types based on the absence or presence of pili
Types T1 and T2 possess pili; virulent forms
Types T3 through T5 devoid of pili; avirulent strains
Protective devices and antigenic variation
o Capsule
o Cell outer membrane proteins
Effects on Ab formation
Leukocyte response
CMI
Protein I major porin protein (Por)
Forms channels for nutrients to pass into and waste products to exit
the cells
Expressed gene: porB protective against inflammatory response
and serum mediated killing
Protein II (Opa = opacity) adherence to phagocytic and epithelial cells
Protein III (Reduction modified protein or RMP) blocks host serum
bactericidal (IgG) action

o Lipooligosaccharides (LOS) or endotoxin

Lipid A moiety and core LOS differentiates it from the LPS
Loosely attached to the underlying peptidoglycan
Major in vivo virulence factor
Mediates damage to body tissues
Elicits inflammatory response
Found in blebs (outer membrane fragments) during rapid growth
Immunoglobulin A protease
o Cleaves IgA on mucosal surfaces

Clinical Infections
Gonorrhea
o Acute pyogenic infection of nonciliated columnar and transitional epithelium
o Primarily acquired by sexual contact
Primary reservoir: asymptomatic carrier
o Means flow of seed
o Often confused with syphilis
o Also called the clap (French: clapoir) meaning brothel
o Incubation period: 2 to 7 days
Acute urethritis
o Men: purulent discharge and dysuria
AHU (Arginine Hypoxanthine Uracil) Asymptomatic men
o Women: cervical discharge, dysuria and lower abdominal pain
Endocervix most common site of infection
If untreated pelvic inflammatory disease (PID) sterility, ectopic
pregnancy, or perihepatitis (Fitz-Hugh-Curtis syndrome)
Blood borne dissemination
o Purulent arthritis, septicemia (hemorrhagic skin eruptions), fever and rash on
the extremities
Anorectal and Oropharyngeal infections
o Common in males who have sex with males; can also occur in women
o Symptoms: pharyngitis, discharge, rectal pain or bloody stools
Ophthalmia Neonatorum in newborns
o Eye infection acquired during vaginal delivery through an infected birth canal
o If untreated blindness
o Treatment: Antibiotic drops or cream 1% silver nitrate drops (Credes
Prophylaxis)
Ocular infections in adults
Laboratory Diagnosis
Specimen Collection and Transport
o Urethra (male), endocervix (female), rectum, pharynx, and joint fluid
o For swabbing: Dacron or rayon swabs
Purulent discharge can be collected directly onto swab
No discharge swab inserted (2-3 cm) to anterior urethra
Rectal culture swab inserted (4-5 cm) to anal canal
o Disinfectants should be avoided in preparing the patient

o Susceptible to drying and temperature changes direct plating of the

specimens within 6 hours

Direct Microscopic Examination

o Gram stains should be prepared from urogenital specimens; not recommended
for pharyngeal specimens (commensal Neisseria spp. can be present)
Gram stain with more than 5 PMNs per field but no bacteria
nongonococcal urethritis
o Symptomatic male: intracellular diplococci appearing as kidney or coffee
bean shape
Presumptive evidence: infection
o Women: not reliable due to presence of saprophytic flora; culture is necessary
o Avirulent forms of the organisms are seen as extracellular

Culture
o Requires the use of Chocolate Agar (primary plating medium) but it also
supports the growth of other organisms
o Media should be warmed to room temperature before inoculation
o Specimens on swab Z pattern and cross-streaked with a loop
o To prevent overgrowth of normal biota and to enhance recovery of species,
selective medium containing inhibitors is used

Selective Media for Isolation of N. gonorrhoeae and N. meningitidis

Selective Medium
Thayer-Martin

Modified Thayer-Martin

Martin-Lewis

New York City

GC-LECT

Inhibitory Agents
Vancomycin
Colistin
Nystatin
Vancomycin
Colistin
Nystatin
Trimethoprim
Vancomycin
Colistin
Anisomycin
Trimethoprim
Vancomycin
Colistin
Amphotericin B
Trimethoprim
Vancomycin
Lincomycin
Colistin
Amphotericin B

Suppressed Organisms
Gram positive
Gram negative
Yeast
Gram positive
Gram negative
Yeast
Swarming Proteus spp.
Gram positive
Gram negative
Yeast
Swarming Proteus spp.
Gram positive
Gram negative
Yeast
Swarming Proteus spp.
Gram positive
Gram positive
Gram negative
Yeast

Trimethoprim

Swarming Proteus spp.

Capnocytophaga spp.

Incubation
o Inoculated plates incubated at 35C, 3-5% CO2 atmosphere
Candle extinction jar (only white wax candles)

Presumptive Identification
o Microscopic Morphology
Gram stain must be performed on all suspected Neisseria isolates
To differentiate Neisseria to Kingella and Acinetobacter spp:
Streak to a plate + 10-unit penicillin disk added
- Edge of the zone of inhibition is stained
o Colony Morphology
Examined daily for growth; held for 72 hours
Appearance: small, gray, transluscent and raised
T1 (moist)and T2(dry): smaller, raised & appear bright in reflected light
T3 to T5: larger, flatter colonies
AHU: smaller
o Oxidase Test
Filter paper or direct plate oxidase test
Oxidase reagent: 1% dimethyl-p-phenylenediamene dihydrochloride or
tetramethyl-p- phenylenediamene dihydrochloride
Fresh isolate: purple color should develop within 10 seconds
Oxidase reagent directly onto a colony: colony turns pink, then black
If subculture is needed: must be done while still in pink; when
black, organism is no longer viable

Definitive Identification
o Carbohydrate Utilization
Traditional method: carbohydrate utilization in cystine trypticase agar
1% of the individual carbohydrate and phenol red (indicator)
Acid (yellow color) is produced in 24-72 hours
N. gonorrhoeae: Glucose only
N. meningitidis: Glucose and Maltose
Rapid Carbohydrate Degradation Tests
Require pure culture but can be read in 2-4 hours
Detect acid production but based on the presence of preformed
enzymes not bacterial growth
Disadvantages: weak acid production from glucose,
misidentification, strains of N. cinerea that give (+) glucose
reactions

Chromogenic Substrates
- detects enzyme production; confirms isolates only from selective media
- Key differentiating factors: use of glucose (N. meningitidis: glucose & maltose)

o Immunologic Assays
Coagglutination
Employ monoclonal antibodies directed against N.
gonorrhoeae attach to kill S. aureus cells (+) agglutination
Fluorescent antibody testing
Uses monoclonal antibodies recognize epitopes on the Por
Extremely sensitive and specific
Confirms the diplococcis morphologic appearance
o Nucleic Acid Assays
Nucleic Acid Hybridization (Nonamplified)
Nonisotopic Chemiluminescent DNA Probe - Hybridizes
specifically with rRNA
Advantage: rapid and sensitive
Disadvantages
Marginally more sensitive than cervical cultures (females)
Used in high risk populations only
Not approved for pharyngeal or rectal specimens
Not used in children or sexual abuse cases
Cannot identify an N. gonorrhoeae infection produced by
-lactamase producing strain
Do not allow for recovery of an organism to be used for
susceptibility testing
Nucleic Acid Amplification (Amplified)
Amplify a specific nucleic acid sequence before detecting the
target sequence with a probe
An extremely sensitive test and do not require viable organisms
Advantage: Less sensitive to transport and storage conditions
Disadvantages
False negative results if the specimen contains inhibitors
Strict attention to procedural factors and quality control
Expensive
o Additional Methods
Superoxol
30% H2O2 and performed in the same way as catalase test
N. gonorrhoeae: immediate vigorous bubbling
N. meningitidis and N. lactamica: weak delayed bubbling
Other Neisseria spp.: weak delayed bubbling or none
Auxotypes
Most common: AHU (arginine, hypoxanthine, uracil)
- High susceptible to penicillin
Labor intensive not recommended for routine clinical
microbiology laboratory
Nonculture Detection Methods
Must not be used in children and cases of suspected sexual assault
or abuse

Antimicrobial Resistance
Type

Acronym

First
Observed

Plasmid-mediated penicillin resistance

PPNG

1976

Chromosome-mediated resistance

CMRNG

1983

TRNG

1985

Plasmid-mediated, high level tetracycline

resistance
Chromosome-mediated spectinomycin
resistance
Chromosome-mediated fluoroquinolone
resistance

QRNG

Mechanism
Plasmid codes for B-lactamases
production
Selection of mutants for low-level
penicillin and tetracycline resistance
Plasmid codes for tetracycline resistance

1981

Mutation causes high level resistance

1991

Mutation causes resistance

PPNG Penicillinase-producing N. gonorrhoeae

CMRNG chromosomally mediated resistant N. gonorrhoeae
TRNG tetracycline resistant N. gonorrhoeae
QRNG quinolone resistant N. gonorrhoeae

Treatment
Cephalosporins currently recommended
Coinfection with C. trachomatis is common
o Primary therapies of N. gonorrhoeae + azithromycin or doxycycline

Neisseria meningitidis

Commensal inhabitant of the upper respiratory tract of carriers

Important etiologic agent of endemic and epidemic meningitis, meningococcemia, and
rarely pneumonia, purulent arthritis or endophthalmitis
Has also been recovered from urogenital and rectal sites oral-genital contact
Meningcoccal carriage involving nonencapsulated strains increase protective
antibody against pathogenic strains

Virulence Factors
Same cellular structural characteristics as N. gonorrhoeae
o Polysaccharide capsule (encapsulated strains)
A worldwide epidemics
B and C community acquired infections
Y meningococcal pneumonia
W-135 invasive disease
o Pili
o Endotoxin
PorA and PorB are both produced
Produce IgA1 protease enzyme that aids in invasiveness

Clinical Infections
Meningitis
o Organism crosses the blood brain barrier and enters the CNS
Acute Meningococcemia
o Asymptomatic carriers
o Pili
o Endotoxin
Purpura hemorrhaging of blood into the skin and mucous membranes
producing bruises
Petechial rash, tiny hemorrhages (skin, lower extremities, joints, lungs
and adrenal glands)
DIC (uncontrollable clotting within the bloodstream)
Hypovolemic shock death
Fulminant meningococcemia
o Hemorrhage into the adrenal glands (Waterhouse-Friderichsen syndrome)

Chronic meningococcemia
o Recurrent episodes of meningococcemia, petechiae formation and development
of arthritis

Laboratory Diagnosis
Specimen Collection and Transport
o CSF, blood, nasopharyngeal swabs and aspirates
o Less common: sputum and urogenital sites
Direct Microscopic Examination
o Gram stain: intracellular and extracellular gram (-) diplococcic
o Encapsulated strains halo
Culture
o Grown on SBA under CO2 bluish-gray colonies
o Chocolate agar small, tan, sometimes mucoid, convex colonies
o For N. lactamica (mimics N. meningitides)
ONPG test is performed to differentiate the 2 species
Identification
o Oxidase and catalse test
o Serogrouping is commonly done by slide agglutination
o -glutamyl aminopeptidase test
(+): N. meningitides
(-): N. gonorrhoeae, N. lactamica and M. catarrhalis
Treatment
Penicillin
Rifampin or Sulfonamide prophylaxis for close contacts
3rd generation cephalosporins patients with meningococcemia
Vaccine: Polysaccharide diphtheria toxoid conjugate vaccine (Menactra)

Moraxella catarrhalis

Branhamella
Genera: Moraxella, Acinetobacter and Psychrobacter
Commensal of the upper respiratory tract

Clinical Infections
Opportunistic pathogen
Upper and lower respiratory infection
Acute otitis media and sinusitis
Life threatening diseases: endocarditis, meningitis, and bacterial tracheitis
Laboratory Diagnosis
Grow on both SBA and CHOC agar
o Smooth, opaque, gray to white colonies, nonhemolytic
o hockey puck remains intact
o (+): oxidase, catalase, DNase & butyrate esterase rxn (Tributyrin - substrate)
Asaccharolytic
Nonpathogenic Neisseria Species
Normal inhabitants of the upper respiratory tract
commensals, saprophytes or nonpathogens
Commonly isolated from the genital tract
Identification
3 Groups
o Group 1: Traditional Pathogens
o Group 2: Commensal Neisseria spp. that can grow on selective medium
o Group 3: Commensal Neisseria spp. that dont usually grow on selective medium
Groups 2 & 3 Saccharolytic (able to metabolize carbohydrates) & Asaccharolytic

Neisseria cinerea

Identical and often misidentified to N. gonorrhoeae

Utilize glucose
Grow on Mueller-Hinton agar but not on TM medium (S-colistin)
Colony morphology: similar to T3 colonies of N. gonorrhoeae
Bacteremia, conjunctivitis, nosocomial pneumonia
Differentiation from N. gonorhoeae: Colistin susceptibility
o N. cinerea - susceptible
o N. gonorrhoeae resistant
Differentiation from M. catarrhalis: reduction of nitrate and (-) DNase rxn
Differentiation from N. flavescens: lack of yellow pigment production

Neisseria flavescens

flavescens = yellow
Yellow pigmented and asaccharolytic

Neisseria lactamica

Commonly found in the nasopharynx of infants and children

Commonly encountered in meningococcal carrier surveys
Only Neisseria spp. that uses lactose
Similar to N. meningitidis
o Colony morphology (slightly smaller)
o Ability to grow on selective media
o Carbohydrate reactions
o Cross-reaction with typing sera
Definitive Test: Lactose utilization or (+) ONPG reaction

Neisseria mucosa

Colonies: large, adherent to the agar and very mucoid

Normal flora of the RT
Isolated from the nasopharynx
Meningitis, endocarditis, cellulitis
Same carbohydrate pattern as N. sicca and N. subflava
o Differs from its ability to reduce nitrite to nitrogen gas, colony morphology and
pigment production

Neisseria polysaccharea

Produces large amounts of extracellular polysaccharide when grown in media

containing 1% or 5% sucrose
Utilize glucose and maltose
Differentiation from N. meningitidis: ability to grow on NA at 35C or production of
polysaccharide on sucrose
Differentiation from N. polysaccharea: growth on SBA or CHOC at 22C and lack of
yellow pigment production

Neisseria sicca

Colonies: dry, wrinkled, adherent and breadcrumb like

sicca = dry (Latin)

Neisseria subflava

Means less yellow

Consists of 3 biovars that differ from one another
Bacteremia, meningitis and septicemia
Clinical description resembles N. meningitidis

Neisseria elongata

Rod-shaped
3 Species: elongata, glycolytica and nitroreducens
Commensals in the upper RT and opportunistic pathogens
To differentiate: catalase test, acid production from glucose and reduction of nitrate
o elongata (-) catalase and nitrate reduction; (+) nitrite reduction

o glycolytica (+) catalase; weak acid producer from glucose

o nitroreduscens (-) catalase; (+) nitrate and nitrite

Neisseria weaveri

Rod-shaped
Normal oral microbiota in dogs
Can be found in humans in infections following dog bites
(+) catalase, reduce nitrite to gas, phenylalanine deaminase (weak)
(-) acid production in any carbohydrates, reduce nitrate
Sensitive to penicillin

Kingella denitrificans

TM1
o Isolated from throat cultures plated on TM medium in carrier subgroups of N.
meningitidis and N. lactamica