Staphylococci

• Gram positive cocci in singly, tetrads, cluster

• Non spore forming
• Facultative anaerobic except S. saccharolyticus.
• Catalase +

Characteristic in Nutrient Agar

• Medium size (pinhead), cream colored, white or rarely light gold.

• “Buttery Looking”
• Golden Yellow pigment

Characteristic in Blood Agar Plate

• Small colony
• Beta Hemolytic species
• Raised Creamy colonies

Micrococci

• Catalase +
• Coagulase negative
• Gram positive cocci
• Found in the environment and skin, mucus membrane microbiota
• Produce cream colored to yellow pigment
• Micrococcus luteus
o Invasive disease of the immunocompromised patient.
 Test
Mannitol Salt Agar
Slide Coagulase Test
Colistin Nalidixic Acid Agar (CNA)
Phenylethyl Alcohol Agar
Catalase Test
Slide Coagulase Test
Glucose Oxidation-Fermentation
Test
Novobiocin Susceptibility Test
DNAse Test
Pyrrolidonyl Arylamidase Test
Microdase Disk (Modified Oxidase)
Description
• Contains 7.5% NaCl
• Carbohydrate: Mannitol
• Indicator: Phenol Red
• Selective Medium for Staphylococcus
• (+): Ferment Mannitol (Yellow)
• (-): Do not Ferment Mannitol (Red)
• isolate Streptococci and Staphylococci
• Colistin: disrupt cell membrane of Gram negative
• Nalidixic: block the DNA
• Inhibits the growth of Gram-negative bacteria.
• To differentiate Staphylococcus and Streptococcus
• Uses 3% Hydrogen Peroxide
• Aerobes and Facultative Anaerobes possess the catalase
• Procedure: Colony + 2 to 3 drops of Hydrogen Peroxide
• (+): Bubbling
• (-): No Bubbling for 30 secs
• To detect Cell bound coagulase/ Clumping Factor
• Fibrinogen converted to Fibrin by Clumping Factor.
• POSITIVE: Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus schleiferi
• (+): White Fibrin Clot (Agglutination)
• (-): Smooth Suspension
• All Staphylococcus ferment glucose except S. saprophyticus, S. hominis, S. xylosus, S. cohnii
• (+): Turn to Yellow
• (-): Green
• Used 5 ug Novobiocin
• Differentiate Staphylococcus saprophyticus and Coagulase Negative Staphylococcus spp.
• Used to detect DNAse activity in aerobic bacteria
• Toluidine blue: Blue → rose pink
• Methyl Green: Green → Colorless
• (+) Clear zone: Staphylococcus aureus
• (-) No clearing: Staphylococcus epidermidis
• To detect Staphylococci producing arylamidase enzyme
• Pyroglutamyl- B naphthylamide + p dimethylaminocinnameldehyde → L-pyrrolidone + B naphthylamine (red)
• (+) Cherry red: S. aureus
• (-) S. lugdunensis, S. intermedius, S. schleiferi
•
• Modified oxidase test to differentiate Micrococcus and Staphylococcus spp.
• Reagent used: 6% tetramethylphenylenediamine hydrochloride in dimethyl sulfoxide)
 • (+): Turn blue in 2 minutes (Micrococcus spp.)
• (-): No Color change

Voges Proskauer (VP) Test • To detect glucose fermentation by Butanediol

Beta lactamase test
Antimicrobial test
• Reagent: Alpha naphthol + KOH
• (+) deep pink/red: S. aureus, S. lugdunensis, S. haemolyticus, S. schleiferi
• (-): S. intermedius
• Cephalosporinase test
Uses cephalosporin or cefinase (with nitrocefin)
(+) pink or red in 60 minutes (10 minutes in other bacteria)
• Acidimetric method
Reagent: citrate buffered penicillin
(+) Red to yellow color
• Iodometric method
Reagent: Phosphate buffered penicillin and starch iodine
(+) Colorless solution
(-) Purple color.
• Vancomycin agar plate
• Oxacillin screen plate
• Cefoxitin disk diffusion: S. aureus and S. lugdunensis
• Double Disk diffusion test
Used of erythromycin and Clindamycin
(+) D zone: flattening at erythromycin of clindamycin zone of inhibition

Gram (+) Cocci

Bacteria Description Virulence Factor Clinical Infection Lab Diagnosis
-Important cause of nosocomial Enterotoxin -Folliculitis Microscopic: Cocci in cluster
infection mainly due to drug -heat stable at 100 Celsius for 30 mins can -Furuncles (AKA Culture Media
resistance cause diarrhea & vomiting. Boils) -BAP: Golden yellow to creamy, beta
-Primary reservoir: Human -Food Poisoning: A, B, D -Carbuncles hemolytic. Buttery looking colonies
naris, Axillae, Vagina, Pharynx Toxic Shock Syndrome Toxin 1 (TSS-1) -Bullous Impetigo -Halophilic: 7.5% - 10% NaCl
& Skin surface -Known as Enterotoxin F -Scalded Skin Coagulase: (+)
-Mode of Transmission: direct Syndrome
-Menstruating (tampon)
contact with unwashed, -Toxic Shock
Exfoliative Toxin
contaminated hands or Syndrome (TSS)
inanimate object. -Epidermolytic toxin -Toxic Epidermal
Staphylococcus -Cause Skin Slough off AKA Ritter Necrolysis (TEN)
aureus disease -Food Poisoning
-Implication of Bullous Impetigo
Cytolytic Toxins
-Hemolysin
 -Alpha hemolysin - damage platelets and
macrophages and cause severe tissue
damage.
-Beta hemolysin (Sphingomyelinase C)-
Acts on the sphingomyelin in the plasma
membrane. AKA ”hot-cold” lysin
-Delta hemolysin – less toxic
-Gamma hemolysin – Associated with
Panton Valentine Leukocidin
Leukocidin
-Panton Valentine Leukocidin

-BAP: white, opaque, small to medium

pinheads and non-hemolytic gray to
Staphylococcus -Hospital Acquired UTI white
epidermidis -Intravascular catheter, CSF -Biochemical test: Coagulase negative,
shunt, prosthetic devices MSA (-)
-Susceptible to novobiocin

-UTI in young, sexually active -BAP: white, opaque slightly larger than
women pinhead, non-hemolytic but some strain
Staphylococcus -Adhere at epithelial cell lining produces yellow.
saprophyticus of urogenital tract. -Urine culture: 10,000 CFU
-Resistant to Novobiocin

-Can mimic S. aureus - Oxacillin resistance

-Mec A gene encodes oxacillin -Catalase: False (+)
Staphylococcus resistance
lugdenensis Endocarditis, septicemia,
meningitis, skin, soft tissue
Staphylococcus -In wounds, bacteremia, -BAP: medium to large with weak
haemolyticus endocarditis, UTI hemolysis
-Vancomycin Resistance
Streptococci PYROGENIC VIRIDANS LACTIC ENTEROCOCCUS

• Gram positive cocci in pairs or chain Temperature 37 45 & 37 10 & 37 10, 45, 37
• Catalase (-)
• Facultative anaerobes Hemolytic Common Beta Alpha And Non- Non- Non-Hemolytic
• Capnophilic (Can stimulate the growth of CO2) Pattern Hemolytic Hemolytic Hemolytic
• Colonies are small and transparent
• Non motile Example S. Pyogenes S. Mutans S. Lactic E. faecalis

Classification

• Bergey’s Classification

o Based on the temperature requirements

Beta hemolysis Alpha, Beta Hemolysis, Non-hemolytic
• Lancefield
o Based on the C substrate of the cell wall (polysaccharides)
PYROGENIC NONPYROGENIC
o Classification is developed by Rebecca Lancefield.
GROUP A GROUP B GROUP C GROUP D
• Brown
o Based on the hemolysis
Streptococcus Streptococcus Streptococcus Enterococcus faecalis
o Presumptive identification, particularly of streptococci and
pyogenes agalactiae dysagalactiae
enterococci. Enterococcus faecium
o α-Hemolysis is partial lysing of erythrocytes in a BAP around Streptococcus equi Streptococcus bovis
and under the colony that results in a green discoloration of the
medium. (S. pnueumoniae)
o β-Hemolysis is complete clearing of erythrocytes in BAP around or under the colonies because of the complete lysis of RBCs.
(S. pyogenes)
o Nonhemolytic, (Gamma) no lysing of hemoglobin. (E. faecalis)
o Alpha Prime - small area of intact RBCs around colony surrounded by a wider zone of complete hemolysis

Gram (+) Cocci

Bacteria Description Virulence Factor Clinical Infection Lab Diagnosis
Lancefield A -M protein – plays a role in Strep throat/pharyngitis- Specimen: Throat swab
adherence and resist (tonsillitis) diagnose through Culture Media
phagocytosis throat swab and direct antigen -SBA in CO2 increase growth
-Lipotechoic Acid, Protein detector. -SBA w/ sulfamethoxazole or colistin or -
F & Fibronectin Binding Impetigo - (Group A) necrotizing -Polymyxin B.
Protein – for the attachment fasciitis; flesh eating bacteria -S. pyogenes at SBA: small, transparent
Streptococcus of streptococcto oral syndrome and smooth with defined Beta hemolysis
pyogenes -Hyaluronic Acid Capsule – -Bacitracin susceptible
prevent opsonization,
 phagocytosis by neutrophil or
macrophages.
-Streptolysin O- anerobic,
measured by Anti
Streptolysin O (ASO test)
-Streptolysin S- hemolysis
when incubate aerobically.
Oxygen Stable
-Streptokinase- convert
plasminogen to plasmin, and
lyse clot
-Hyaluronidase- AKA
spreading factor
- Enzyme that solubilize the
ground substance of
mammalian connective
tissue.
---Exotoxin –Stimulate T
cell proliferation.
Erysipelas- dermal layer skin -SXT (sulfamethoxazole) Resistant-
infectious; painful swelling antibiotic test
reddish spot -PYR Hydrolysis Positive
Cellulitis- redness and -Immunologic test: ASO, Anti-Dnase,
accumulation of fluid (pamumula Antistreptokinase, Antihyaluronidase
at pamamaga)
Scarlet fever- (strawberry color
tongue= caused by exotoxin;
susceptibility test Dick test (show
of redness of the skin)
Necrotizing fasciitis- chronic
impetigo
Streptococcal toxic shock- STS;
condition whole organ system
shut down and lead to death; due
to toxin
Rheumatic Fever- Damage heart
valves
Acute glomerulonephritis
-Occur after pharyngitis
-Complement, inflammatory
response cause damage to the
glomeruli

Streptococcus -Group B (acid stable
agalactiae polysaccharide at cell wall)
-9 capsular polysaccharide
serotype
-Microbiota of female genital
tract and lower GIT
-Contains residue of sialic acid
-Capsule
-Hemolysin- for hemolytic
pattern (beta hemolytic)
-CAMP - unique virulence
factor, known as Christie–
Atkins–Munch, Peterson;
help us to identify if its group
B streptococci or group A
streptococci
-Neuraminidase
-Dnase
-Hyaluronidase
-Mastitis
-Disease on Newborn
--Early stage: Pneumonia &
sepsis
--Late stage: Meningitis & sepsis-
have a normal flora of vagina
-Healthy woman become ill after
childbirth, abortion
-Elderly with serious condition.
-SBA: Grayish, white mucoid colonies in
-Beta hemolysis
-Specimen: vagina/rectal swab
-Todd Hewitt w/ Colistin & Nalidixic
acid
- (+) CAMP & Hippurate test
Agglutination test
-Resistant for SXT and Bacitracin
-Strep B Carrot Broth
- (+) Beta hemolysis

Streptococcus -member of the S. mitis group -Lobar pneumonia -Microscopic: cocci in pairs that are
pneumoniae -Common in Bacterial meningitis -Meningitis common in elderly lancet shaped
(pneumococcus) -Causative agent of lobar and children due to the otitis -Optochin Test- Antibiotic that interferes
pneumonia media with the ATPase and production of ATP.
-Otitis media (+)/Susceptible: With zone of
--In patient under 3 years old inhibition or clearing
 -C substance, which is similar to
the C carbohydrate of the various
Lancefield groups
-The capsule is antigenic and
identify by antisera in a Neufeld
Quellung test.
Enterococcus -group D
-Natural inhabitant of intestinal
tract of human and animals
-Mainly non hemolytic and alpha
hemolytic.
-Exhibit pseudocatalase
-Can grow in extreme conditions:
Bile, 6.5% NaCl, 45C and
Alkaline pH
-Hemolytic Uremic Syndrome (-)/Resistant: No zone of Inhibition
-Bile Solubility- Test that differentiates
Streptococcus pneumoniae from other
Alpha hemolytic Streptococci
--Reagent: 2% (in test tube) to 10%
Sodium desoxycholate (slide),
--Lysis depends on the presence of an
intracellular autolytic (Amidase)
--(+): Lysing of bile: clear
--(-): Bile not lyse: Turbid
-Neufeld Quellung test -Capsule swell
after the addition of anticapsular serum
-E. faecalis- Extracellular -Specimen: Blood, Urine, Wound
surface adhesin proteins, -Culture Media: Trypticase soy, BHI
extracellular serine protease, supplemented w/ 5% sheep Blood
and gelatinase -Has ability to produce acid in
--Two subunit toxin called carbohydrate broth
cytolysin -Hydrolyze Arginine
-Common cause of UTI due -Tolerance of 0.04% tellurite
to catheterization or urologic -Utilization of pyruvate
manipulation. -Produce acid from methyl-α-D-
-Endocarditis glucopyranoside,
-growth around 100-µg efrotomycin acid
disk
-Motility
-Bile Esculin Test- identification of
Enterococcus vs. Streptococcus bovis
--Reagent: Bile and Esculin
--(+) Blackening of Agar: Enterococcus
faecalis
--(-) No color change
-6.5% NaCl Test or Salt Tolerance Test
--ability of the
organism to grow in high concentration.
--Differentiate Enterococci from
Nonenterococci
--Reagent: Brain heart infusion broth +
6.5% NaCl + Glucose + Bromcresol
purple
--(+) Turbidity w/ or w/o color change
(purple)
--(-) without turbidity and no color
change
Streptococcus Streptococcus like Organism Streptococcus like Streptococcus like Organism Streptococcus like Organism
like Organism Organism
Aerococcus -Airborne -Vancomycin susceptible
-Resemble to viridans
streptococci but microscopically
similar to Staphylococci

Gemella -Similar to viridans streptococci

Lactococcus -Group N Streptococci

-Similar to enterococci but do not
produce acid from carbohydrates

Leuconostoc -Similar biochemical -BE Hydrolysis (+),

characteristics with Enterococci -can grow on 6.5% NaCl.
and viridans. -PYR and LAP (-)
-Seen in vegetable, plant surface
and milk products

Pedicoccus -Resistant to vancomycin

-BE and LAP (+), PYR (-)
 Streptococcus pyogenes and Streptococcus agalactiae Identification
Test
Leucine Aminopeptidase Test (LAP)
Bacitracin Susceptibility Test
Hippurate Hydrolysis
CAMP Test
(Christie, Atkins, Munch-Petersen)
Pyrrolidonyl-Alpha Naphthylamide Hydrolysis
6.5% NaCl Test or Salt Tolerance Test
Description
• Presumptive identification of Catalase Negative Gram + Cocci
• Detection of Leucine Aminopeptidase
• Leucinebeta-naphthylamide + leucine aminopeptidase+
cinnamaldehyde --- beta-nephthylamine (red color)
• (+) Development of red color
• (-) No color change or slightly yellow
• Presumptive identification of Streptococcus pyogenes in Throat
swab
• Inoculate at blood agar with SMZ and bacitracin disk.
• Susceptible: Streptococcus pyogenes
• Resistant: Streptococcus agalactiae
• To differentiate Streptococcus agalactiae and other Beta Hemolytic
Streptococci
• Reagent: Ninhydrin, Hippurate Disk
• Hippurate+ H2O---- Benzoic Acid+ Glycine
• (+): Purple (Streptococcus agalactiae)
• (-): Colorless or Yellow (Streptococcus pyogenes)
• Use beta lysin producing strain of Staphylococcus aureus
• Synergistic hemolysis between group B
Streptococcus and beta-hemolytic
Staphylococcus aureus
• (+) Enhanced hemolysis in arrowhead pattern (e.g., Streptococcus
agalactiae)
• (-) No arrowhead shown
• Presumptive of Beta hemolytic
• Streptococci and non-hemolytic D.
• (+): Red color
o Streptococcus pyogenes, Enterococcus, Aerococcus,
Gemella
• (-): Yellow
• ability of the organism to grow in high concentration
• Differentiate Enterococci from Nonenterococci
• Reagent: Brain heart infusion broth + 6.5% NaCl + Glucose +
Bromcresol purple
• (+) Turbidity w/ or w/o color change (purple)
• (-) without turbidity and no color change
 Neiserria spp.
• Aerobic, Nonmotile, Gram negative diplococci
• All are diplococci except N. elongata, N. weaver
and N. bacilliformis that are rod shape.
• All species are catalase (+), cytochrome oxidase (+)
except: N. elongata and N. baciliformis
• Capnophilic (Can stimulate the growth of CO2)
• Natural habitat: Mucous membrane of RT & UGT.
• N. gonorrhea and N. meningitides are human
pathogen
• N. weaver are commensal in URT of dogs.
Neisseria gonorrheae Transport System
• James E Martin Biological Environmental
Chamber (JEMBEC)
• Gonopak
• Transgrow
• If direct plating is impossible: Amies medium w/
charcoal (6 hours)
Oxidase Test
Carbohydrate Utilization test
Multitest Method
Virulence Factor
• Pili or Fimbrae
o Exhibits hairline projection
o T1 and T2 possess pili while T3 to T5 do not have pili
• Lipooligosaccharide or Endotoxin
o Lipid A moiety
o Core LOS
o Factor that mediates damage to body tissue and elicits inflammatory response
o Contains blebs
• Immunoglobulin A protease- commonly seen in body fluids.
• Porin Protein
o Channel for nutrient to pass into waste product exit the cell.
o Por A & B for N. meningitidis, Por B: N. gonorhhea
• Protein II (opacity)
o Facilitate adherence of phagocytosis and epithelial cells
• Protein III (reduction modified protein, Rmp)
o Blocks the host serum bactericidal action.
Neisseria gonorrheae Culture Media
• Chocolate Agar is media of choice
• Thayer Martin- isovitalex and antibiotic (vancomycin)
• Modified Thayer Martin- trimethoprim lactate= inhibit swarming
• Martin Lewis- Anisomycin
• New York City- Amphotericin, STD disease (Mycroplasma hominis, ureaplasma,
urealytica)
• GC LECT- Lincomycin
• Reagent used: 1% dimethyl-p-phenylenediamine dihydrochloride & tetramethyl-p-
phenylenediamine dihydrochloride
• (+): Purple color/ blue color
• Agar used: Cystine
Trypticase Agar
• Indicator: Phenol red
• (+) Red
• (-) Yellow
• Enzyme method (substrate + biochemistry test), ELISA
• Also identify Haemophilus
 Immunologic Assay
Matrix Assisted Laser Desorption/ Ionization time of
flight Mass Spectrophotometry
Commensal Neisseria spp.
• Coagglutination (use monoclonal antibody against N. gonorrhea)
• Fluorescent Antibody Test
• No cross rxn: N. cinerea but w/ cross rxn: N. lactamica
• Superoxol test- 20-30% H2O2 (+)- extreme bubbling
• Protein signatures and based on size & charge on electric field
• DNase (-)
• NAAT= PCR
• Normal inhabitants
o Rarely cause disease as opportunistic infections
Groups
• Group 1: pathogens
• Group 2: commensals that grow on selective media
• Group 3: commensals that do not grow on selective media

Gramm (-) cocci

Bacteria Description Virulence Factor Clinical Infection Lab Diagnosis

Neisseria -acute pyogenic infection of
gonorrheae noncilliated columnar and
transitional epithelium
-Acquired thru sexual contact
-Occur in urethra,
endocervix, anal canal,
phatrynx, conjunctiva
- “Flow of seed”
-Secondary cause of STD.
-Men are symptomatic while
women are asymptomatic
-SPS inhibits N. gonorrheae
(Gelatin is added)
Neisseria -Commensal and invasive
meningitidis -Site: Rectal and urogenital
-Highest incidence found on
the infants
-Adhere at the
nasopharyngeal mucosa
-Meningococcemia/ sepsis
-Pili (T1-T2 virulent, T3- T5
avirulent)
--Exchange of genetic material
--Attachment to human mucosal cell
invasion
--Inhibits phagocytosis
-Capsule, Lipooligosaccharide
-Protein II (Opa)
--Adhere to phagocytic and
epithelial cell
-Protein II (RMP)
--Block bactericidal effect of IgG
-Outer membrane Porin (PorB)
--Protection from the host immune
response
-Newborn: Ophthalmia neonatorum:
gonococcal eye infection during
vaginal delivery
-Acute purulent urethritis, prostatitis,
and epididymitis: Males
-Acute cervicitis: Females
-pharyngitis, anorectal infections,
conjunctivitis
-Pelvic inflammatory disease (PID)
may cause sterility ectopic
pregnancy or perihepatitis also referred
to as Fitz-Hugh–Curtis syndrome.
-Microscopy: Gram negative diplococci,
coffee or kidney shaped with presence of
PMN or pus cells.
--Pharyngeal specimen unnecessary to do
gram stain
-->5 PMN: Non gonococcal urethritis
--If direct plating is impossible: Amies
medium w/ charcoal (6 hours)
-Gram stain of CSF: Cytocentrifuge
-SBA and CHOC agar (72 hours)
-SBA: Medium size, gray and convex,
encapsulated strains are mucoid and green
tinge
-CHOC: Colorless to gray, convex colonies
-Glucose & Maltose (+)
 --Occur without meningitis
-Disseminated Intravascular
Coagulation
-Septic Shock
-Waterhouse Friderichsen
Syndrome (Hemmorhage of
the Adrenal Gland)
Increase in Defieciency in C5
& C8
Moraxella -Family Moraxellaceae
catarrhalis -Opportunistic pathogen of
the URT
-Can cause LRTI in adults
with chronic obstructive
pulmonary disease
-Predisposing factor: Age,
Immunodeficiency,
Neutropenia and chronic
debilitating disease
Non sporeforming, Non- Branching, Catalase Positive Bacilli
• Corynebacterium spp.
• Arcanobacteria
• Rhodococcus
• Listeria, Erysipelothrix, Gardnerella
Tinsdale Agar or Cystine Tellurite Agar
-Rapid O-nitrophenyl-B D
galactopyranoside
-Detects Lactose utilization
-Gamma Glutamyl Aminopeptidase (+)
-Produce Beta Lactamase -Cause of Otitis media and sinusitis -SBA and CHOC: smooth opaque, gray to
-Predisposing factors -Pneumonia white colonies
--Advanced age -Sinusitis and otitis media - “Hockey puck”
--Immunodeficiency --Third most common cause in children -Can tolerate 28 Celsius
----Can have severe infections -Rarely: endocarditis, meningitis, and -Inhibited on gonococcal agar with colistin
--Neutropenia tracheitis -Dnase (+) – halo clearing
-Specimen collection and -Butyrate esterase (+)- tributyltin test
identification (blue+)
--Middle ear effusion -resistant to penicillin
--Nasopharynx
--Sinus aspirates, sputum, or
bronchial aspirates
-Identification
--Oxidase and catalase positive
--Asaccharolytic
--Positive DNase and butyrate
esterase reactions
Sporeforming, Non-Branching, Catalase Positive Bacilli
• Bacillus spp
o Bacillus subtilis
o Bacillus anthracis
o Bacillus cereus
• (+) Black to brownish colonies
• Corynebacterium: Brown halo surrounding colonies

Biochemical Test: UREASE TEST (Christensen’s Method)
Biochemical Test: Nitrate reduction Test
Additional Test
o C. diphtheria, C. ulcerans, C. pseudotuberculosis, Staphylococcus,
Streptococcus
• Used to determine the ability of the organism to produce urease
• Reagents: Urea, Phenol red (indicator)
• Urea (pH 6.8) + Urease → Ammonia + CO2 (pH 8.1)
o (+): Pink or magenta pink (Rapid: w/in 24 hours & Weak: After several
days. (C. ulcerans and C. pseudotuberculosis)
• (-): No color change/light orange (C. diphtheriae)
• Test to determine the ability to reduce nitrate to nitrite
• Nitrate + Sulfanilic acid + Alpha naphthylamine → diazonium salt (RED) + N2O2
• Zinc to confirm nitrate is not reduce
• (+): Red/no color change
• (-) No color change/Red
• Glucose Fermentation (+)
• Maltose Fermentation (+)
• No gas production

Bacteria Description
Corynebacterium -Microbiota of Skin, Mucous
spp. membrane of Human and
Animal
-2 types of Corynebacteria
--Nonlipophilic
--Lipophilic
---Fastidious, grow for 48
hours or more
---Culture media must have
Lipids like Tween 80 or
Serum (C. jeikeium)
Corynebacterium -Also known as Kleb-
diphtheriae Loeffler Bacillus
-Significant pathogen of
Corynebacterium spp
- “Chinese Character”
Corynebacterium -Skin Microbiota
amycolatum
Corynebacterium -Name after Johnson & Kaye
jeikeium -Skin Microbiota
Corynebacterium -Nasopharynx Microbiota
pseudodiphthericum
Corynebacterium -Veterinary pathogens
pseudotuberculosis
Aerobic Gram (+) Bacilli
Virulence Factor
-Diphtheria Toxin
--lysogenic Beta phage w/c carry
tox gene
--Also produce by Corynebacterium
ulcerans & Corynebacterium
pseudotuberculosis
-Composed of 2 fragments
--A: Cytotixic and disrupt protein
synthesis
--B: Binds to the receptors on the
cell.
-Produce dermonecrotic toxin
Clinical Infection
-Cause of Diphtheria
-URT ((Respiratory Diphtheria)
--Common site: Tonsil, Pharynx
--Pseudomembranous Colitis (“Bull’s
Neck”)
-Cutaneous diphtheria
--Nonhealing ulcer with dirty gray
membrane
Treatment:
-Administration of Antitoxin
-Penicillin
-Erythromycin
-Diphtheria vaccine
-Can cause Endocarditis and Prosthetic
joint infection
-Assoc. w/ prosthetic valve
endocarditis, septicemia, meningitis
-Respiratory infection in
immunocompromised patient
-Cause granulomatosus lymphadenitis
Lab Diagnosis
-Gram Stain: Slightly curved gram-
positive rods with unparallel side
--Slightly wide ends producing “club
shape”
--Significant pathogen:
Corynebacterium diphtheriae
-Microscope: Pallisade (Picket Fence),
Snapping form, Barbed dumbbell, Club
shaped
-Microscopic: Stain with Methylene
Blue, Albert Stain, Neisser’s Stain,
Ponder’s Stain
--Babes Ernst Granules= Metachromatic
granules due to accumulation of
polyphosphate. Reserve nutrients
-Culture Medium
-SBA: Small zone of Beta hemolysis
-Loeffer’s serum: Use to visualize
metachromatic granules
-Tinsdale Agar: Black to Brown due to
reduction of tellurite
Corynebacterium -Skin & Nasopharynx -Can cause Endocarditis and
striatum Microbiota Prosthetic joint infection
-Device related infection
Corynebacterium -Produce diphtheria toxin -Mastitis -Nitrate reduction (-)
ulcerans -Diphtheria like illness
Rothia spp. -Rothia dentocariosa -Endocarditis (R. d) --Microscopic: coryneform bacilli,
--Normal oral flora -Bacteremia (R. m) forming short gram-positive bacilli but
-Rothia mucilaginos -Endocarditis (R. m) also branching filaments
-Wound infections (R. m) --Broth: Coccoid cells
-Eye infections (R. m) -Nitrate-positive
-Nonmotile
-Esculin hydrolysis-positive
-Urease-negative
Listeria -Nonsporeforming, -Listeriolysin O -Listeriosis - Catalase positive Bacilli
monocytogenes Nonbranching --Hemolysin that damage the -CNS infection -Microscopy: Gram positive
-Isolated in crustaceans, flies phagosome membrane preventing -Affected thru eating of coleslaw, coccobacilli but become coccoidal when
and ticks killing of organism by macrophage chicken, hotdog, luncheon meat subculture and variable from older
-Ability to survive with -Catalase & Superoxide dismutase -Drug of choice: Ampicillin, Penicillin, culture (Not seen in CSF)
phagocytes -Phospholipase C Aminoglycoside, Macrolides -Culture: SBA: Narrow growth of Beta
-Surface protein hemolysis (resembles S. agalactiae)
--Induces phagocytosis by increase --Small, round, smooth, translucent
adhesion and penetration to cell. --Can grow in 0.5 to 45 Celsius
--Grows at 4 Celsius (Cold Enrichment)
Erysipelothrix -Commensal pathogen and Other Test: -Erysipeloid- Localized skin disease -Catalase negative
rhusiopathiae can be opportunistic • Catalase (-) --Resembles to streptococcal -Resistant to Vancomycin
pathogen. • Nonmotile erysipelas -Microscope: Thin, rod gram positive
-Mode of transmission: • Aerobic or facultatively --Septicemia assoc. with septicemia with long filaments Singly, short chains
Through cuts or scratches anaerobic gram + rod. --Alcohol abuse with previous heart in V shape. Appear in variable
-Resistant to salting, pickling • Urease (-) disease -Culture: Nutrient broth with 1%
and smoking • VP (-) -Cutaneous infection Glucose incubated in 5% CO2
-Predisposed individuals: • Hydrolysis of Esculin (-) -Note: Mainly Self Limiting --SBA (24 hours): Nonhemolytic,
Fish handlers and the • H2S (+) -Drug of Choice: Penicillin pinpoint, smooth form is transparent,
veterinarians glistening and convex with entire edge
--Becomes alpha hemolytic after few
days
-Gelatin Stab Test: E. rhusiopathiae
(+): Test Tube Brush like pattern/ pipe
cleaner (22 Celsius)
Arcanobacterium -A. haemolyticum -Narrow zone β-hemolysis -Catalase negative
--Pharyngitis and pruritic --Black dot on agar surface under - Lipase and lecithinase positive
scarlatiniform rash with the colony - Exhibits a CAMP inhibition reaction
desquamation of the skin of
the hands and feet
-A. pyogenes
--Sepsis and wound
infections in those with
animal contact
-A. bernardiae
--Bacteriemia, wound
infections, urinary tract
infections (UTIs), and septic
arthritis
Gardnerella -Normal biota of Urogenital -Cause UTI on men and women -Microscopic: Short pleomorphic,
vaginalis tract -Treatment: Metronidazole, gram + rods (sometimes variable)
-Decrease Lactobacillus, Clindamycin -Grow with 5% to 7% CO2
Increase pH -SBA: pinpoint nonhemolytic colonies
-Also known as Clue cells -CM: Human Blood Bilayer Tween
(HBT)= Contains Human Blood, Beta
hemolytic small gray and opaque
-Catalase (-)
-Oxidase (-)
-Hippurate Hydrolysis (+)
-Whiff test: Vaginal secretion + 10%
KOH → Fishy amine odor
Sporeforming, NonBranching, Catalase Positive Bacilli (Gram (+) Bacilli)

Bacillus spp. -Other Bacilli are -Grow at SBA but not in CNA
Thermophiles (55 Celsius)
(Bacillus stearothermophilu)
-Non pigmented
-Has endospores on aerobic
and anaerobic
Bacillus anthracis -Also known as “Woolsorter -D-Glutamic acid Capsule - Disease of livestock -Microscopy: Large, square ended,
disease”, Rag picker’s --Resistant to hydrolysis of enzymes -3 Forms of Anthrax gram positive
Disease --Protect the organism from --Cutaneous- Characterized by Black --Unstained central spores that gives a
phagocytosis. eschar (small dark area appears in the “Bamboo rods appearance”
--Antibody against capsule does not center of a ring but no pus) -Endospore stain- Spores: Green,
confer immunity Vegetative cells: Red
 -Anthrax Toxin
--Protective Antigen- Binding of
EF and LF. Permits the binding to
specific receptor
--Edema Factor- Adenylate Cyclase
that increase the cyclic Adenosine
Monophosphate in host cell
--Lethal Factor- Protease that kill
host cells by disrupting the
transducting of extracellular
regulatory signals
Bacillus cereus -Common cause of food -Diarrheal Enterotoxin
poisoning --Incubation >6hrs. Diarrhea, lasts
-Opportunistic on susceptible 20-36hrs.
host -Emetic Enterotoxin
--Incubate ,6hrs, severe vomiting,
lasts 8-10hrs.
Aerobic -Morphologically resemble Nocardia can be identified using
Actinomycetes: fungi paraffin bait test.
Nocardia Species --Exhibit wrinkled, chalk like --Only Nocardia can break it down
and orange tan as a substrate.
-Slow growers, may take 3 to
6 days
--Inhalation/Pulmonary
(Woolsorter’s disease- Spores is
inhaled into the pulmonary
parenchyma
--Gastrointestinal Anthrax- Spores
inoculated into a lesion on the
intestional mucosa after ingestion.
Abdominal pain, Nausea, anorexia,
vomiting Bloody diarrhea.
-Drugs: Fluoroquinolone
-Treatment: Clindamycin and/or
Amnioglycoside.
-Resistant to penicillin
-Susceptible to sulfonamides
-Capsule: using India Ink
-Culture: SBA: Nonhemolytic, large,
gray flat with an irregular margin,
filamentous (“Medusa Head)”
-Phenylethyl Alcohol Medium
--For stool sample, Weak growth
-Polymyxin-Lysozyme –EDTA-
Thallous Acetate (PLET)
--For contaminated samples
-Bicarbonate Agar
-Agar with Penicillin (0.05 – 0.5
U/mL)
--Large spherical bacilli (microscope)
--“String of Pearls”
-Catalase (+)
-Non motile
-Ferment Glucose but do not ferment
Mannitol, Arabinose, Xylose
-Lecithinase (+): Egg Agar
-Grow at 7% NaCl
-Grow as low as < pH 6
-Susceptible to Penicillin
-Culture- stool must have atleast 10^5
cell per gram
-Resistant to penicillin & Betalactam
except carbapenems.
-Motility test: Positive
-Penicillin (R)
-Lecithinase (+)
-String of pearls: (-)
-Penicillin Agar, PEA (+)
-Gelatin Hydrolysin (+)
-Sulfur granules
-Wet mounts: Crush granules to see
cellular morphology
-Microscopy: Gram-positive
branching filaments are seen in direct
smears from sputum or aspirated
material.
 -Rare but high mortality rate --May show beading appearance
(40%) --May take 3 to 6 days to grow on
media

N. cyriacigeorgica -Confluent -No “sulfur granules”

and N. farcinica bronchopneumonia
-Necrosis with abscesses

N. brasiliensis -Cutaneous infection termed “Sulfur granules” present

actinomycotic mycetomas
-Trauma to skin- Major destruction of
the tissue and bone

Diarrheal Emetic
TEST B. anthracis B. cereus

Type Hemolysin, Cytotoxin, Acid Resistant

Nonhemolytic Growth at SBA Nonhemolytic Beta hemolysis

Medusa Head “Frosted glass appearance”

Incubation 8-16 hours 1 – 5 hours

Motility (-) (+)

Duration 12 to 24 hours 6-24 hours

Food Meat, soup, vegetables, Fried or boiled rice Penicillin S R

pudding, sauces
Lecithinase (+) (+)
Stable to heat (-) (+)

String of Pearl (+) (-)

Fluid accumulation on (+) (-)
rabbit ileal segment
Penicillin Agar (-) (+)
Stimulate Adenylate (+) (-)
cyclase in intestinal
epith cell PEA (-) (+)

Lethal for mice (+) (-) Gelatin Hydrolysis (-) (+)

Response Diarrhea Vomiting

Identification of Nocardia and Similar Species

• Methods of identification
o Substrate hydrolysis (casein, tyrosine, xanthine, and hypoxanthine)
o Other substrate and carbohydrate utilization (arylsulfatase, gelatin liquefaction, and carbohydrate utilization)
o Antimicrobial susceptibility profile
o Fatty acid analysis by high-performance liquid chromatography

Other Actinomycetes

• Actinomadura
o Actinomadura madurae and Actinomadura pelletieri
o Similar to Nocardia
• Streptomyces
o Saprophytes
o Streptomyces somaliensis is an established human pathogen associated with actinomycotic mycetoma in many countries
o Streptomyces anulatus (formerly Streptomyces griseus) has been increasingly isolated from many clinical specimens
• Gordonia
o aerobic, catalase-positive, gram-positive to gram-variable, partially acid-fast, and nonmotile
o Nocardioform
o weak acid fastness and the absence of arylsulfatase
o Reduce nitrate and the absence of mycelia.
• Tsukamurella
o gram-positive, aerobic, catalase-positive, and partially acid-fast.
o 16S rRNA gene sequencing and fatty acid analysis
• Rhodococcus
o SBA, the colonies resemble Klebsiella and can form a salmonpink pigment on prolonged incubation
o not ferment carbohydrates and shows a variable reaction to many characteristics
• Tropheryma whipplei
o Whipple’s disease
o best made by microscopic examination of endoscopic biopsy specimens.
o periodic acid–Schiff staining
Hacek
H- Haemophilus spp.
A- Aggregatibacter actinomycetecomitans
C- Cardiobacterium hominis
E- Eikenella corrodens
K- Kingella Spp.
HACEK

• Opportunistic pathogens
• Dysgonic (slow and poor growing) on BAP and CAP 7 to 14 days incubation
• Mainly heart valve that cause endocarditis
• Both vegetative and nonfermentative bacilli
• Utilize aminolevulinic acid
• Normal microbiota of oral cavity

HEMOPHILUS SPP

• From the Greek word

o Haima- Blood
o Philos- Love
• Obligate bacteria on the mucuos membrane of human
• Inhibits at the URT except H. ducreyi (eyes)
• Fastidious, non-motile, capnophilic, facultative anaerobes
• Cannot grow on pure BAP
o X factor (Hemin)
o V factor (Nicotinamide adenine dinucleotide)
 • Human pathogen
o H. influenza
o H. ducreyi
o H. parainfluenzae
o H. parahemolyticus
o H. aegypticus
o H. segnis (Aggregatibacter segnis)
GROWTH FACTOR

• X Factor
o Hemin
o Found at the RBC

• V Factor
o Nicotinamide Adenine Dinucleotide
o Do not grow on SBA
o Lysing of RBC to readily available the V factor.
o Satellitism
▪ Organism like S.aureus, S. pneuominae and Neisseria produce V factor as a by
product
HEMOPHILUS INFLUENZAE

• Common name: Pfeiffer’s Bacillus

• Associated with influenza
• Only second invader of influenza
• Typeable form
o Capsular characteristic (A to F)
o Type B cause serious infections in humans and cause meningitis in unvaccinated children.
• Non Typeable form
o Part of the URT
o 2nd agent of otitis media
• Virulence Factor
o Capsule
▪ 6 antigen type (a to F)
▪ Encapsulated strain (Hib)
o IgA Protease
▪ Inhibits the IgA of the RT
o Adherence by Fimbrae
o Does not produce endotoxin
• Clinical Infection
o Encapsulated strain
▪ Septicemia
▪ Meningitis
▪ Arthritis
▪ Epiglottis
▪ Tracheitis
 ▪ Pneumonia
o Non typeable Hi
▪ Conjunctivitis
▪ Sinusitis
▪ Otitis media
▪ Pneumonia
▪ Bacteremia
HAEMOPHILUS AEGYPTICUS

• Common name: Koch-Weeks Bacillus

• Observe in conjunctivitis exudates from Egyptians
• Commonly refer as “pinkeye”
Haemophilus influenza biogroup aegypticus

• Cause conjunctivitis in pediatric population

• Non encapsulated
• Brazilian purpuric fever (BPF) in Brazil in 1984
HAEMOPHILUS DUCREYI

• Causative agent of chancroid/soft chancre

• Genital ulcer disease (GUD)
• Not part of microbiota
• Common site of infection: Penis, Labia, or Vagina
• Suppurative (pus forming)
• Buboes (inguinal lymph nodes)
OTHER HAEMOPHILUS SPP

• Haemophilus parainfluenzae
o Found in the oral cavity
o Causative agent of endocarditis
o After a routine dental procedure
o Primary site of infection: Mitral Valve
• Haemophilus parahaemolyticus
o Cause pharyngitis
LABORATORY DIAGNOSIS
Specimen Processing

• Blood, CSF, middle ear exudate, joint fluids, upper and lower respiratory tract specimens,
swabs from conjuctivae, vaginal swabs, and abscess drainage
• Note genital specimens (moisted w/ phosphate buffer saline) for H. ducreyi is highly
fastidious
• Direct plating is preferred
MICROSCOPIC ANALYSIS

• Small, gram-negative coccobacilli to long filaments

• Capsules of H. influenza: direct smears as clear, nonstaining areas (“halos”) surrounding the
organisms in purulent secretions.
• Acridine orange or methylene blue stain of the specimen may help in detecting Haemophilus.
HAEMOPHILUS DUCREYI

• School fish, Rail road tracks.

• Finger print arrangement
CULTURE: CHOC Agar

• Haemophilus ducreyi: small, flat, smooth, nonmucoid, transparent to opaque colonies or appears
tan or yellow on CHOC agar
• H. parainfluenzae colonies appear tannish and drier with a medium to large size compared with H.
influenza.
• Haemophilus infleunzae: appear translucent, tannish, moist, smooth, and convex, with a distinct
“mousy” or beachlike odor
BLOOD AGAR PLATE

• Uses Horse or Rabbit Blood

• H. influenza: Nonhemolytic
• H. parahaemolytic: Betahemolytic
SATELLITISM

• Organism like S. aureus, S. pnuemoniae and Neisseria produce V factor as a by product

• Examples:
o Haemophilus influenza
o All Heamophilus spp. that are clinically significant except H. ducreyi
X & V FACTOR

• Haemophilus Quad Plate

o Media with X factor only, with V factor only, with X and V factors, and with X and V
factors with horse red blood cells.
PORPHYRIN TEST

• Alternative method for differentiating the heme-producing species of Haemophilus.

• Reagent: Kovac’s Reagents (p-dimethylaminobenzaldehyde)
• Delta-aminolevulinic acid (ALA) →
• Porphobilinogen + Kovacs’ → RED COLOR → Ultraviolet light (wood’s lamp) → REDDISH
ORANGE in 360 nm (Porphyrin)
Aggregatibacter aphrophilus

• Greek aphros and philia: foam loving or desiring high concentration of CO2.
• Cause of endocarditis
 • Found in dental plaque and gingival scrapings
• Colonies are convex, granular, and yellow with an opaque zone near the center on CHOC agar.
• Has X factor dependent and independent strain.
• Positive for Maltose, Sucrose, Glucose, Lactose (only lactate fermenter organism)
Aggregatibacter actinomycetecomitans

• Formerly known in the genus Actinobacillus.

• Isolated with Actinomyces
• Major virulence factors: collagenase and leukotoxin
• Catalase (+)
• Urase, indole, esculin, citrate (+)
• A distinctive “star shape with four to six points” in the center of the colonies is often seen at 48
hours.
• At broth: granular and adhere wo the side of tube
• Glucose (+) with or without gas
• Do not ferment Xylose, mannitol, maltose, lactose, sucrose
Cardiobacterium hominis

• Normal microbiota of the nose, mouth and throat and may be present in the GIT
• False gram-positive reaction
• Organisms tend to form “rosettes”, swelling, long filaments, or sticklike structures in yeast extract.
• On agar “pitting” may be produced
• C. hominis fragments glucose, mannitol, sucrose, and maltose but not lactose
• Oxidase and indole- positive
• Negative for urease, nitrate, gelatin, and esculin.
Eikenella corrodens

• “Corroding bacilli”
• Normal biota of the oral and bowel cavities
• Results of trauma after human bites or fights
• Cause of adult periodontitis, pneumonia, osteomyelitis, arthritis, and postoperative tissue
infections.
• Pitting or corrode the surface of the agar
• Nonhemolytic on SBA, a slight greening effect or yellow secondary to growth may occur around
the colonies
• Chlorine bleach like odor from the agar surface
• Broth: adhere to the sides of the tube and produce granules
• Lysine carboxylase (+)
• Arginine dihydrolase (-)
Kingella Spp.

• They tend to resist decolorization in the gram stain

• Coccobacillary to short bacilli with squared ends that occur in pairs or short chains
• Oxidase (+)
 • Catalase (-)
• Can grow on Neisseria selective agar particularly K. denitrificans
• K. denitrificans is positive for glucose fermentation and nitrate reduction and might grow at 42C
Kingella kingae

• K. kingae: gram- negative short coccoid bacilli

• K. kingae weakly ferments glucose and maltose but is negative for sucrose.
• Produce a yello- brown pigment
• A spreading, corroding colony or a smooth, convex, and β-hemolytic colony
Capnocytophaga spp.

• Family Flavobacteriaceae and includes bacteria previously called DF- 1 and DF- 2 (dysgonic
fermenters)
• They are thin and often fusiform (pointed ends) resembling Fusobacterium spp.: spindle- shaped,
coccoid, and curved filaments
• C.canimorsus and C. cynodegmi: Dog bites
• “Gliding motility on solid surface” (No flagella)
• Produce a yellow- orange pigment; they can resemble colonies of E. corrodens but spreading from
the center
• Capnocytophaga spp. ferment sucrose, glucose, maltose, and lactose
• Reduce nitrates (+) and hydrolyze esculin

Zoonotic Bacteria
Brucella Spp.
• Zoonotic disease (Category B)
• AKA Bang Bacillus
• Obligate aerobe
• Causes Undulant fever, Malta fever, Crimean and Mediterranean
• Common in patient with exposure to animals
• Direct person-to-person transmission
• Four species that are ost commonly associated with huan illness are B. melitensis (goat or sheep)
[biosafety level III], B. abortus (cattles), B. suis (pigs/ swine), and B. canis (dogs)
• New: B. ovis, and B. neotomae
• Can cause spontaneous abortion to animals because it localized in erythritol rich area.
Microscopic
• Gram negative nonmotile, may appear as coccobacilli or bacilli with sandy appearance
• Facultative intracellular pathogens that can reside within phagocytic cells.
Culture
• Blood agar: small, convex, transluscent and brownish as age
• Thayer- Martin or Martin- Lewis media from contaminated specimens
o Thayer-Martin usually uses in Neisseria spp.
• B. abortus requires niacin/ nicotinic acid but inhibited by theonine dye
• Medium: Castaneda Bottles, Trypticase soy agar
Other test
 • Rapid Urease (2 hours)
• Catalase positive
• Oxidase positive
• Serum agglutination (> 1: 160 titer)

Species Natural host H2S (lead Urease Fushin Theonine

acetate metd.)
B. abortus Cattles + + - +
B. canis Gog - + + -
B. melitensis Goats/ sheep - V - -
B. suis swine + + + -

Francisella tularensis
• Causes Tularemia
• Strictly anaerobic
• Rabbit fever, deerfly fever, lemming fever, and water rat tappers’ diseases
• Contracted through ingestion, inhalation, arthropod bite (e.g., ticks, biting flies), or contact with
infected tissues
• Common clinical form is ulceroglandular (enlargement of the lymph nodes)
• Category A
Microscopic
• Small, nonmotile non- sporeforming, gram- negative bacilli or coccoid bacteria
• Facultative intracellular pathogens.
Culture Media and Incubation
• Slow grow rate and not visible before 48 hours.
• Require supplementation with cysteine, cystine, or thiosulfate for growth on successive passage.
• CHOC, modified Thayer-Martin, and buffered charcoal yeast extract (BYCE) agars and Mueller
Hinton and tryptic soy broths may be used.
• Morphology: Gray white raise colony with smooth appearance
• Plates should be checked daily for 14 days
Other test
• Oxidase, urease, satellite or X and V test (-)
• Weakly positive for catalase and β- lactamase activity (inhibits some antibiotics)
• Presumptive identification:
o Direct fluorescent antibody (DFA)
o PCR
o Slide agglutination
o Single serology test: 4 fold increase in antibody titer

Pasteurella spp.
Pasteurella multocida
• Zoonosis
• Often resides in respiratory tract and oral cavity of birds and mammals
• Three subspecies: multocida, septica, gallicida
• Bipolar staining (safety pin appearance when the poles of the cells are more intensely stained)
 • Catalase (+) and oxidase (+) and ferment glucose with weak to moderate
• SBA and CHOC: grayish colonies nonhemolytic
• Produce a narrow green-to brown halo around the colony after 48 hours
• “Mushroom smell”
Pasteurella bettyae
• Obtained from placenta, amniotic fluid, blood, rectal sites, abscesses, and urogenital specimens
• Can grow in MacConkey Agar (MAC)
• Glucose and Fructose (+)
• Catalase (+)
• Oxidase (variation)
• Indole (+)

Legionella Spp.
Legionella pneumophilia
• Legionnaire’s disease
o Febrile disease with pneumonia
o Epidemiological bacteria
• Pontaic fever
o Febrile disease without pulmonary involvement
• Host factor
o Suppressed immune system, chronic lung disease, alcoholism, and heavy smoking,
predispose individuals to legionnaires’ disease.
• Virulence factor
o Ability to enter, survive, and multiply within the host’s cells, especially bronchoalveolar
macrophages (facultative intracellular)
o Produce proteolytic enzymes.
LEGIONENELLA SPP.

• L.longbeachae
o Associated with exposure to gardening materials, such as compost and potting soil
• Leginonella spp. can tolerate chlorine concentrations of 3 mg/Dl
• Cold water systems, ornamental fountains, whirlpool spas, humidifiers, respiratory theraphy
equipment, and industrial process waters
• Multiply over 20*C to 43*C and survive for varying periods at 40* C to 60* C
SPECIMEN COLLECTION AND HANDLING

• Sputum, bronchoalveolar lavage, and bronchial washings

• Saline or buffer should not be used
• Urine is an important specimen to be collected for antigen detection (for L. pneumophila
serogroup 1)
MICROSCOPIC EXAMINATION

• Pleomorphic, weakly staining, gram-negative bacilli

• Counterstain: safranin can extend to at least 10 minutes
• L. micdadei:
o Weak acid fast (in kinyoun staining)
ISOLATION METHOD

• Acid temperature to eliminate contamination particularly in sputum

• Diluted specimen 1:10 with 0.2 N KCI- HCI and allowed to stand for 5 minutes. (Acid treatment)
• Fastidious, aerobic bacteria that do not grow on SBA and require L-cysteine for growth
• Buffer charcoal yeast extract with L-cysteine
o Selective: Polymyxin B, anisomycin and vancomycin or cefamandole
BCYE: BUFFER CHARCOAL YEAST EXTRACT

• A “ground-glass” appearance, light gray and granular

• Periphery of the colony: pink or light blue or bottle green bands with a furrowed appearance
• Illuminated with a long-wave ultraviolet light (366 nm)
LEGIONELLA SPP.
PHENOTYPIC CHARCTERISTICS/BIOCHEMICAL TEST

• Slow growth (3 to 5 days)

• Characteristics of “ground-glass” colony morphology
• Lightly staining, gram-negative bacillus
• Requires L-cysteine for primary isolation
• No growth on unsupplemented sheep blood agar (SBA)
• Asaccharolytic
• Catalase or oxidase: weakly positive
OTHER TEST

• URINE ANTIGEN TEST

o Radioimmunoassay, microplate enzyme immunoassay, and rapid
immunochromatographic assay for Legionella antigen
o L. pneumophlia serogroup 1

• DIRECT FLUORESCENT ANTIBODY TEST

o For Legionella spp in the lower tract
o Fluorescein isothiocyanate-labeled conjugates
o Appear as bright yellow to green, short or coccobacillary bacilli with intsense peripheral
staining

• DNA DETECTION
o Probe-Tec ET Legionella
o Nucleic Acid Amplification test

• INDIRECT FLUORESCENT ANTIBODY (IFA) ASSAY

o Heat-killed or formalin-killed bacteria are fixed to a microscope slide
o Less cross reactivity reaction
▪ Cross rxn: Mycoplasma and Chlamydia
o A fourfold increase in IFA titer to at least 1:128 from the acute serum phase
BORDETELLA SPP.

• Species
o B. parapertussis
o B. pertussis
o B. bronchiseptica
o B. avium
o B. hinzii
o B. holmesii
o B. petrii
o B. trematum
• Whooping cough or pertussis B. pertussis and B. parapertussis
GENERAL CHARACTERISTICS

• Small gram negative bacilli or coccobacilli

• Obligate aerobic bacteria
• Do not ferment carbohydrates
• Oxidize amino acids
• Inactive in biochemical test systems
• Catalase (+)
• Less fastidious can grow on MAC
B. PARAPERTUSSIS AND B. PERTUSSIS
VIRULENCE FACTOR

• Filamentous hemagglutinin (FHA)

o Facilitate attachment to ciliated epithelial cells
• Pertussis toxin (PT)
o A protein exotoxin that produces a wide variety of responses in vivo
o Modification of host proteins by adenosine diphosphate-ribosyl transferase, which
interferes with signal transduction
• Adenylate cyclase toxin
o Inhibits host epithelial and immune effector cells by inducing supraphysiologic
concentrations of cyclic adenosine monophosphate
• Tracheal cytotoxin
o Causing ciliostatis, inhibiting DNA synthesis, and promoting cell death
WHOOPING COUGH: PHASES

• Catarrhal phase
o Insidious and nonspecific and include sneezing, mild cough, runny nose, and perhaps
conjunctivitis, although infants can develop apnea or respiratory distress or both
o Highly contagious
• Paroxysmal phase
o Whoop of the end of the coughing spell
• Convalescent phase
o Decrease in frequency and severity of the coughing spell
OTHER BORDETELLA SPP

• Bordetella brochiseptica
o RT pathogen of dogs
o Also cause kneel cough
o Can cause infection in immunosuppressed patient that has exposure to animals
o Cause pertussis syndrome
LABORATORY DIAGNOSIS

• Polymerase chain reaction

• Direct fluorescent antibody
• Note organism cannot survive outside the host
SPECIMEN COLLECTION

• Nasopharyngeal Aspirates or Swab

o Uses calcium alginates or dacron polyester
o Also uses flocked swabs (higher sensitivity)
o Directly plating is required
o Swab can be express in 1% casein hydrolysate (casamino acid)
o Transport media: amies with charcoal for 24 hours delayed of specimen
• Throat swab
o Discourage to be used because of lack of sensitivity
CULTURE MEDIA

• Regan Lowe Transport Media

o Half-strength charcoal agar containing 10% horse blood and 40 mg/ dL cephalexin
o For overnight transport
Enterobacteriaceae

• Facultative anaerobes
• Non sporeforming
• Gram negative bacilli
• Motile at 35 Celsius except: Klebsiella, Shigella & Yersinia (motile at room temp or in cold temp)
• Non encapsulated except: Klebsiella & Enterobacter
• Ferment glucose
• Reduce Nitrate to nitrite
• Oxidase negative except Plesiomonas shigelloides
Virulence Factor

• Adherence: prevent being removed from tissue

• Toxins: food poisoning
• Invasive enzymes: spreading factors
• Antibiotic resistance
o Extended spectrum β-Lactamases (ESBL)
• Serology
o Somatic O antigen (somatic antigen)
▪ Heat stable antigen located in the cell wall (LPS)
▪ Found at the cell wall
▪ E. coli and Shigella
o Flagellar H antigen
▪ Flagellar antigen; heat labile
o Capsular K antigen
▪ Capsular antigen; heat labile
▪ K1 antigen of E. coli, Vi antigen of Salmonella typhi
Group of Enterobacteriaceae

• Opportunistic pathogens
o Intestinal Microbiota of animals and man
o Do not initiate disease but can cause extraintestinal infection outside the normal body site
(UTI, Sepsis, Meningitis etc.)
o Escherichia coli, Citrobacter, Enterobacter, Klebsiella, Proteus, & Serratia
• Overt or True Pathogen
o Acquire thru ingestion of contaminated food and water.
o Very significant when found in specimen
▪ Salmonella spp., Shigella spp., Yersinia spp.
Escherichia coli

• “Colon Bacilli”
• Microbiota of the Large intestine (Colon)
• Leading cause of UTI
• Has both Sex pili and adhesive fimbriae
Uropathogenic Escherichia Coli

• Produce factors that allow them to attach to the urinary epithelial mucosa.
• Virulence Factors
o Pili: adhere to epithelial cells and not be washed out with urine flow.
o Cytolysin: Can kill immune effector cells and inhibit phagocytosis and chemotaxis of
certain white blood cells
o Aerobactins: chelate Iron.
• Disease: UTI, Acute Pyelonephritis
Gastrointestinal Pathogens

• Based on virulence factors, clinical manifestation, epidemiology, and different O and H serotypes
o enterotoxigenic Escherichia coli (ETEC)
▪ Occur at proximal small intestine
▪ Refer as Traveller’s Diarrhea
▪ Consumption of contaminated food or water.
▪ High risk for patient with achlorhydria
▪ Virulence Factor
• Heat Labile toxin (cholera like toxin)
o Accumulation of cAMP
o Hyper secretion of fluids
• Heat Stable Toxin
o Stimulate guanylate cyclase to increase production of cyclic
guanosine monophosphate
o Hypersecretion of fluid
o enteroinvasive Escherichia coli (EIEC)
▪ Produce dysentery or Shigella like
▪ Watery diarrhea with WBC
▪ Mode of Transmission: person to person by fecal-oral route
• detect invasiveness using monolayer cell cultures with human epithelial-2
(HEp-2) cells.
▪ Virulence Factor: Invasin
o enteropathogenic Escherichia coli (EPEC)
▪ Infantile diarrhea (1940) (For patient less than 1 year old)
▪ Stools mainly with mucus but no blood.
▪ Virulence factor: H antigen, Intimin
o enterohemorrhagic Escherichia coli (EHEC) [E coli O157:H7]
▪ Associated with hemorrhagic diarrhea, colitis, Hemolytic uremic syndrome.
▪ Hemolytic Uremic Syndrome- low platelet count, hemolytic anemia, and kidney
failure.
▪ Virulence Factor:
• Verotoxin 1- a phage-encoded cytotoxin identical to the Shiga toxin (Stx)
produced by Shigella dysenteriae type I.
o Damages Vero cells (African green monkey kidney cells)
o Toxin neutralized by Shiga toxin antibodies
• Verotoxin II- Immunological different from STx and Verotoxin I.
 o Enteroadherent Escherichia coli
• Diffusely adherent Escherichia coli (DAEC)
• Enteroaggregative Escherichia coli (EAEC).
▪ Associated with watery diarrhea and UTI
▪ Uropathogenic DAEC strains are closely associated with cystitis in children and
acute pyelonephritis in pregnant women.
▪ Adhering to the surface of the intestinal mucosa and Hep 2 cells
• “Stack brick pattern” cell by means of fimbrae
Escherichia hermanii

• Yellow-pigmented
o Isolated from cerebrospinal fluid (CSF), wounds, and blood
o Isolated from foodstuffs such as raw milk and beef
• Clinical significance still not fully established
E. vulneris

• Newest member (Wound infections)

4-methylumbelliferyl-β-D-glucuronide (MUG) assay

• A biochemical test used to screen isolates for E. coli O157:H7.

• E. coli O157:H7 do not produces the enzyme β-glucuronidase.
• Negative: No fluorescence (O:157H7)
• Positive: With fluorescence
Other Test to Detect E. coli O157:H7

• Enzyme linked Immunosorbent Assay

• Latex Agglutination: O157, H7
• Sorbitol fermentation: Sorbitol (-), Sorbitol MacConkey Agar: Colorless
• Detecting Verotoxin- Fourfold increase in verotoxin neutralizing antibody titer.
• EMB: Green Metallic Sheen
• MAC: Pink (Lactose Fermenter)
Biochemical test

• Fermentation of glucose, lactose, trehalose, and xylose

• Indole (+)
• Methyl Red (+) and Voges-Proskauer (-)
• H2S, DNase, urease, or phenylalanine deaminase (-)
• Citrate (-)
Klebsiella species

• Usually found at the GIT of humans and animals

• Species: K. pneumoniae subsp. pneumoniae, K. oxytoca, K. pneumoniae subsp. ozaenae, K.
pneumoniae subsp. rhinoscleromatis, K. ornitholytica, K. planticola, and K. terrigena.
• MAC: exhibits mucoid pink color colonies.
 Test for Klebsiella pneumoniae
Klebsiella pneumoniae subsp. Pneumoniae • MAC: Pink Mucoid colonies
• Common isolates of Klebsiella sp. • Differential Test: (+) String Test
• “Friedlander’s Bacillus” • Neufeld Quellung test: Positive
• Causative agent of community acquired pneumonia • IMVIC Reaction: - - ++
• “Currant jelly like” sputum • Weak urease positive
• Virulence factor: Polysaccharide capsule • TSI: A/A, gas (+), H2S (-)

Other Klebsiella spp.

• Klebsiella oxytoca
o Similar to K. pneumoniae
o Indole (+), Ornithine (+), Alginate (-)
o IMVIC: + - - + +
• K. pneumoniae subsp. ozaenae
o Nasal secretion and cerebral abscesses.
o Cause Atrophic rhinitis
• K. pneumoniae subsp. Rhinoscleromatis
o Rhinoscleroma- Intense swelling and malformation of the entire face and neck
o Africa and South America
Raoutella Species

• R. ornithinolytica
o Indole and ornithine decarboxylase positive
• R. planticola
o Both species found in urine, respiratory tracts, and blood
Enterobacter species

• Resembles with Klebsiella when growing on MAC

• Significant organism
• Enterobacter aerogenes
• Enterobacter cloacae
o Arginine dihydrolase (-)
o Urease weak (+)
o Lysine decarboxylase (-)
• Enterobacter gergoviae
o Found in respiratory samples but rare at blood samples
• Enterobacter cancerogenus (formerly E. taylorae)
o associated with osteomyelitis after traumatic wounds
Biochemical Test

• Ornithine decarboxylase (+), KCN (+)

• Lysine decarbosylase (+): Except E. cloacae and E. gergoviae
• Sorbitol Fermentation (+): E. aerogenes and E. cloacae
• Urease & Malonate (+): E. cloacae
• IMVic Rxn: - - + +, TSI: A/A, gas (+), H2S (-)
Cronobacter sakazakii

• Known before as Enterobacter

• produces a yellow pigment
• causing meningitis and bacteremia
• coming from powdered infant formula
• Biochemical test: IMVIC similar to Enterobacter species
o BHI Agar: Yellow pigmentation
Pantoea agglomerans

• Causes nosocomial outbreak of septicemia due to contaminated IV fluid.

• Biochemical Test
o Triple decarboxylase (-)
o MAC: Clear colonies
o IMVIC: - v + v
o TSI: K/A Gas (-) H2S (-)
Serratia species

• Opportunistic pathogen
• Resistant to wide range of antibiotics.
• S. marcescens, S. liquefaciens, S. rubidaea, S. Plymuthica: Pink to red colonies at 25 Celsius due
to prodigiosin pigment
• Serratia marcescens
o Late lactose fermenters
o Gelatinase, Urease, ONPG (+)
o Arabinose (+)
• Serratia ordofera
o “Rotten potato like odor”
• Serratia liquefaciens
o Ferment Arabinose
o KCN (+)
• Biochemical Test
o IMVic Rxn: - - + +
o TSI: K/A Gas (+=) H2S (-)
o Dnase, Lipase, ONPG, Gelatinase (+)
Proteus species

• Isolated from urine, wound and ear infections.

• Can cause Acute glomerulonephritis in patient with UTI and catheterization.
• Rapid urease producer
• Culture & Biochemical Test
o MAC: NonLactose fermenter
o Exhibit Swarming
o Burnt chocolate or burn t gunpowder odor
o Phenyalanine Deaminase (=)
o LIA: R/A
 • Proteus mirabilis • Proteus vulgaris
o Urease activity can lead to kidney stones
o Produce swarming in BAP o Indole (+)
o Indole (-) o Ornithine decarboxylase (-)
o Ornithine decarboxylase test (+) o IMViC: + + - v
o IMViC: - + v v o K/A Gas (+) H2S (+)
o K/A Gas (+) H2S (+)
Providencia spp

• Nosocmial outbreaks involving burn units.

• P retgerri (+) and stuartii (-) can be differentiate into urease test
o P. alcalifaciens, P. stuartii, P. rettgeri, P. rustigianii, and P. heimbachae. P. rettgeri
• Biochemical Test
o MAC: NonLactose Fermenters
o Phenylalanine Deaminase Test (+)
o IMViC: + + - +
o LIA: R/A
o TSI: K/A Gas (-) H2S (-)
• Providencia rettgeri
o Can cause UTI
o Diarrheal disease among travelers
o Resistance to antibiotic
• Providencia stuartii
o Nosocomial outbreaks in burn units
o Providencia alcalifaciens
o Feces of children with diarrhea
Morganella species

• Can cause UTI and neonatal sepsis

• Motile but do not swarm
• Biochemical test
o MAC: Non Lactose Fermeter
o PAD test (+), KCN, Urease, Ornithine decarboxylase (+)
o IMVIC: + + - -
o LIA: R/A
o TSI: K/A Gas (+) H2S (-)
Citrobacter spp.

• Before under the Salmonelleae

• Biochemical test resembles to Salmonella (false + in Salmonella agglutination test) but Mac
resembles to E. coli
• Biochemical Test
o Slow urease
o Slow Lactose Fermenters
o MAC: Lactose fermenter resembles pink colonies
 • Citrobacter freundii
o Extraintestinal pathogen
o UTI, pneumonia and intraabdominal abscesses, Endocarditis in IV drug user
o Produce group 1 cephalosporinase
o IMVic: - + -+
o TSI: A/A or K/A Gas (+) H2S (+)
o KCN and ONPG (+)
• Citrobacter koseri
o Cause neonatal meningitis and brain abscess in nursery units
o IMViC: + + - +
o TSI: K/A Gas (+) H2S (-)
o KCN and ONPG (-)
Edwardsiella species

• E. tarda, E. hoshinae, and E. ictaluri.

• Edwarsiella hoshinae
• Edwardsiella tarda
o Isolated from snakes, birds and water
o Recognized human pathogen
• Edwarsiella ictaluri
• Biochemical Test
o Causes enteric septicemia in fish
o MAC: Non Lactose Fermenters
o Urease (-)
o Lysine Decarboxylase (+)
o IMViC: + + - -
o TSI: K/A Gas (+) H2S (+)
Salmonella species

• typically found in cold-blooded animals as well as in rodents and birds, which serve as their natural
hosts. (Salmonella enterica subsp. Enterica serotype Typhi)
• Not part of the Large intestinal microbiota
• Mode of Acquisition: Ingestion of contaminated food.
• Species: S. enterica (the type species of the genus), S. bongori
• S. enterica subsp. enterica (also called subspecies I),
• Salmonella serotype Typhi,
o Salmonella serotype Choleraesuis,
o Salmonella serotype Paratyphi
o S. enterica subsp. salamae (subspecies II)
• S. enterica subsp. arizonae (subspecies IIIa): before Arizona
• S. enterica subsp. diarizonae (subspecies IIIb)
• S. enterica subsp. houtenae (subspecies IV)
• S. enterica subsp. indica
• Virulence Factor
o Fimbrae, Enterotoxin
o Ability to transverse intestinal mucosa.
• Antigenic Structure
o Somatic O - lipopolysaccharide located in the outer membrane of the cell wall
o Flagellar H - Phase 1: Homologous antisera & Phase 2: Heterologous antisera
o Vi antigen (Salmonella serotype Typhi) – preventing phagocytosis
 • Biochemical Test
o Hektoen Enteric Agar & XLD: clear, colorless, non–lactosefermenting colonies; colonies
with black centers
o MAC: Non Lactose fermenter
o All Salmonella are motile except Salmonella serotype Pullorum and Gallarum
o All produce gas except Gallinarum and Typhi
o LDC & H2S (+) except S. serotype Paratyphi
o Urease (-), KCN (-)
o IMViC:
▪ -+-+
▪ - + - - (Salmonella sserotype Typhi
o TSI: K/A Gas (+) H2S (+)
• Clinical Infection
o Gastroenteritis (Salmonellosis)
▪ Cause by Salmonella enterica subsp. Enterica
▪ Source: Poultry products, milk and handling of pets
▪ Infective dose 10 ^6
▪ Nauseae, Fever, Chills, wtery diarrhea and abdominal pain
o Enteric fever (Typhoid fever)
▪ Febrile disease after eating contaminated food prepare by a carrier or individual
▪ Salmonella enterica subsp enterica serotype Typhi
▪ “Rose spots” during the second week of infection
o Bacteremia
▪ Occurs with or without extraintestinal infection caused by Non typhoidal
Salmonella sp.
▪ Causative agents
• Salmonella serotype Typhimurium
• Salmonella serotype Paratyphi
• Salmonella serotype Cholerasuis
Shigella species • Serogroup
o Serogroup A: Shigella dysenteriae
• Not member of GI Tract
o Serogroup B : Shigella flexneri
• Closely related to Escherichia coli.
o Serogroup C Shigella boydii
• Non motile, Intracellular pathogens o Serogroup D: Shigella sonnei
• Reservoir: Human • Antigenic Structure
• Most the species can cause bacilliary dysentery o Somatic O
• Culture & Biochemical Test o Some posses K antigen
o MAC: Colonies are clear and colorless
Nonlactose Fermenter
o SSA: Colorless without black centers
o IMViC: v + - -
o TSI: K/A Gas (-) H2S (-)
o All do not produce gas except S. flexneri
o All are mannitol fermenter except S. dysenteriae
o All do not decarboxylate lysine
o All do not decarboxylate ornithine except S. sonnei
 • Shigella dysenteriae • Shigella sonnei
o Most virulent Shigella species o Self limiting
o Virulence factor: Shiga toxin o Characterized by watery diarrhea
o Urease (-), LDC (-)
Bacilliary dysentery

• Cause by Shigella dysenteriae type 1

• Characterized by Acute inflammatory colitis & Bloody diarrhea
• Infective dose <200 bacilli
• In children can cause Rectal Prolapse
• Tramistted via fecal oral or by flies, fingers, food, feces.
Yersinia pestis

• “Plague bacillus”
• Bioterrosim Agent
• Transmitted thru bite of the infected flea.
• Causetive agent of bubonic plague
• Vector: Xenopsylla cheopis or Oriental rat flea.
• Virulence factor: Endotoxin, Coagulase and fibrinolysin
• Biochemical Test
o MAC: Clear and Colorless Non Lactose Fermenter
o BAP: Pinpoint at 24 hours
o Broth: Colonies have a “Stalactite pattern”
o TSI: K/A Gas (-) H2S (-)
• Yersinia enterocolitica
o Causative agent of enterocolitis or waterborne gastroenteritis
o Isolated form packed RBC
o Survive at the cold temperature
o Mode of acquisitions: Undercooked food, dairy products, handling of pets
o Reservoirs: Rabbits, swines, dogs, cow
o Medium & Biochemical Test
▪ Cefsulodin –Irgasan – Novobiocin: Bull’s eye appearance, dark red, burgundy
center
▪ MAC: Colorless, Non Lactose Fermenter
▪ IMViC: v + - -
▪ TSI: K/A Gas (-) H2S (-)
• Yersinia pseudotuberculosis
o Pathogen of rodents (guinea pig)
o Motile at 18 to 25 Celsius
o Mode of acquisition: Direct contact with infected animals
o Reservoir: Farm and domestic animals (birds)
o Biochemical
▪ MAC: Colorless, Non Lactose Fermenter
▪ Urease & rhamnose (=)
▪ TSI: K/A Gas (-) H2S (-)
Plesiomoas shigelloides

• Part of human microbiota

• Cause secretory diarrhea
• Cross agglutinate with shigella
• Only oxidase (+) in Enterobacteriaceae
• Mode of acquisitions: Ingestion of undercooked seafood (oysters and shrimps)
Culture and Biochemical test

• MAC: Colonies are clear and colorless (NLF)

• BAP: Shiny, opaque smooth and non hemolytic
• HEA growth
• Inositol-brilliant green labile salt agar for enhancement of plesiomonads from specimen
• Oxidase (+)
• Decarboxylase test: Positive trio decarboxylase
• Inositol: Positive
• ImVic: + + - -
• TSI: K/A Gas (-) H2S (-)