REVIEW
OF
CLINICAL
MICROBIOLOGY
PART II
MJ Weintraub, DVM
Prokaryotes Bacteria
• Cell wall/envelope structure
– GN, GP, AF & wall-less
• Genome
• Morphology, arrangements, capsule/slime layer, flagella,
fimbriae, pilus
• Horizontal gene transfer mechanisms
• 70s ribosome
• 1 Circular, double stranded DNA chromosome/genome
• Plasmids: extrachromosomal, circular, dsDNA, self-replicating
Eukaryotes
Fungi, Protozoa & Helminths
•
•
•
•
Organelles
80s ribosome
Genome in nucleus
Protozoa
– flagella, cilia, pseudopods or non-motile
– Some cysts, trophozoites
• Fungi
–
–
–
–
Dimorphism
Chitin in cell wall
Ergosterols plasma membranes
Some capsules
• Helminths
– Developmental stages if any
Viruses & Prions
• Viruses
–
–
–
–
–
Intracellular obligate parasites
Acellular agent
RNA or DNA genomes
Capsid &/or envelope
Bacteriophages
• Prions
– Infectious pieces of protein
Common Stains - Bacteria
• Gram
– Understand technical errors
– GN & GP
– Endospores – clear within cell
• Acid Fast
– AF – red & non-AF – blue
– Ziehl Neelsen (heat)
– Kinyoun (cold-detergent)
• Acridine Orange - fluochrome
– Bacteria difficult id in a specimen – blood
COCCI
–
–
–
–
Single or pairs (diplococci)
Chains (streptococci)
Clusters (staphylococci)
tetrads
BACILLI
• vary size & length
• Ends may be square or rounded
• Long & filamentous
• Coccobacilli -very short
• Fusiform -tapered or pointed ends
• Curved
• Pleomorphic -species varies shape within pure culture
• Single or chains or palisades
SPIROCHETES -helical turns
– Vary in length & number
Common Stains - Bacteria
• Methylene Blue
– Metachromatic granules Corynebacterium diphtheriae
– AF counterstain
• Endospore
– Red/pink endospore green-living cell
Common Stains - Fungi
• Lactophenol Cotton Blue (LCB)
– Medically important fungi
• Calcofluor White – fluorochrome
– Fungi – bright apple-green or blue-white
• India ink
– Cryptococcus neoformans capsule
Types of Growth Media
• Minimal media
– Contents are simple
– Not usually used in clinical microbiology lab
• Nutrient media
– Complex and made of extracts of meat or soy beans
– Ex. Nutrient broth, Trypticase Soy Broth, TSA agar
• Enriched media
– Contains added growth factors, such as blood,
vitamins, and yeast extract
– Ex. Blood agar & Chocolate agar
Types of Media
•
Selective media
– Contains additives that inhibit growth of some bacteria but supports others to grow
– MacConkey, CNA, Mannitol Salt
•
Differential media
– See metabolic differences between groups or species of bacteria
– MacConkey, Mannitol Salt
•
Transport medium
– Holding medium
– Preserves viability of microorganisms in specimen but does not allow multiplication
– Ex. Stuart broth, Cary-Blair
Media
Category
Used For
Sheep Blood Agar
Enriched
Good for fastidious organisms, most organisms will grow,
interpret hemolysis
Chocolate
Very Enriched
Very Enriched Good for highly fastidious organisms,
Phenylethyl Alcohol Agar Selective Selective for GPC
Haemophilus
Maconkey Agar
Selective & Differential
Selective for GN, differential LF and NLF
(Enterobacteriaceae & oth GNRs)
Maconkey-Sorbitol Agar
(SMAC)
Selective & Differential
E. coli O157:H7, differential for sorbitol fermentation
Eosin-Methylene Blue
Agar
(EMB)
Selective & Differential
Selective for GNRs, differential for lactose & sucrose
fermentation
(Enterobacteriaceae & oth GNRs)
Hektoen Enteric Agar
(HE)
Highly Selective &
Differential
Selective for enteric pathogens, differential for sucrose &
lactose fermentation
H2S
Enhances growth of Salmonella & Shigella
Xylose-Lysine
Desoxycholate Agar
(XLD)
Highly Selective &
Differential
Selective & GNR & enhances growth of Shigella
differential for sucrose, lactose & xylose fermentation
H2S & lysine (decarboxylation)
Used to ID Salmonella & Shigella
Media
Category
Used For
Colistin & Nalidixic Acid Blood
Agar (Columbia CNA)
Selective
Selective for GPC, read hemolysis
Phenylethyl Alcohol agar
Selective
Selective for GPC
Modified Thayer Martin
Selective & Enriched
Neisseria gonorrhoeae (vancomycin, colistin, nystatin,
thrimethoprim)
Martin Lewis
Selective & Enriched
Neisseria gonorrhoeae (vancomycin, colistin, nystatin,
thrimethoprim)
New York City agar (NYC)
Selective & Enriched
Neisseria gonorrhoeae, Ureaplasma urealyticum &
Mycoplasma hominis
Campylobacter Blood Agar
(CAMPY)
Selective & Enriched
Campylobacter spp.
Cefsulodin-Irgasan-Novobiocin
(CIN)
Selective
Yersinia enterocolitica
Thiosulfate Citrate Bile Sucrose
Salts (TCBS)
Selective &
Differential
Vibrio spp. Differentiates Vibrio spp. based on sucrose
fermentation
Media
Used For
Category
Buffered Charcoal-Yeast
Extract Agar (BCYE)
Enriched
Legionella spp.
Fletcher Semisolid Medium
Enriched
Leptospira spp.
Loeffler Coagulated Serum
Slant
Enriched
Clostridium diphtheriae
Tellurite Blood Agar with
or/without Cystine
Tinsdale Agar
Selective, differential &
enriched
Selective and differential
Corynebacterium diphtheriae
Clostridium diphtheriae
Bordet-Gengou Blood Agar
Selective and enriched
Bordetella pertussis and B. parapertussis
Reagan-Lowe Medium
Selective
Bordetella pertussis
Mannitol Salt Agar
Selective and differential
Selective for staphylococci and differential
for mannitol fermentation
Bile Esculin Agar
Selective and differential
Selective for Group D Strep and
Entercoccus spp.
Media
Category
Used For
Blood Agar – Anaerobe
Enriched
Fastidious
Kanamycin & Vancomycin Laked
Blood Agar (KVLB)
Selective & Enriched
Bacteroides spp. & Prevotella spp.
Bacteroides Bile Esculin agar
(BBE)
Selective & Differential
Bacteroides fragilis group
Phenylethyl Alcohol agar
Anaerobic
Selective & Enriched
Anaerobes
Egg Yolk agar (EYA)
Differential
Clostridium spp. Lecithinase, lipase & protease activity
Cylcoserine Cefoxitin Fructose
agar (CCFA)
Selective & Differential
Clostridium difficile
Thioglycolate Broth (THIO)
All purpose media
Back-up broth
Todd Hewitt Broth & LIM broth
Selective
Group B streptococci
Chopped Meat Broth
Selenite Broth
anaerobic
Cultivation of anaerobes
Enrichment
Salmonella spp. & Shigella spp.
bacteria
• JEMBEC transport system
– Neisseria gonorrhoeae
– Sodium bicarbonate & sodium citrate
– Inhibition of contaminating bacteria and fungi
Environmental Factors
• pH
– Most pathogenic bacteria grow best at a neutral pH
– media 7.0 – 7.5
• Temperature
– Categories
• Psychrophiles – optimal growth 10 to 20O C
• Mesophiles – optimal growth 20 to 40O C
• Thermophiles – optimal growth 50 to 60O C
Atmospheric requirements
• Obligate aerobe
– require oxygen for growth
• Aerotolerant anaerobe
– can survive in presence of O2 but do not use O2 in metabolism
• Obligate anaerobe
– cannot grow in presence of oxygen
• Facultative anaerobe
– grow either with or without oxygen
– Most opportunistic pathogens
• Capnophilic
– Best at increased CO2
– Capnophilic bacteria need 5% -10% CO2
• Microaerophilic
– reduced level of oxygen to grow
• Ex. Campylobacter spp. – requires 5% to 6% O2
Fermentation & Respiration
Bacteria use biochemical pathways to catabolize CH2O & produce energy
• Respiration (Oxidation)
– efficient energy-generating process
– Obligate aerobes & facultative anaerobes
• Fermentation
– Anaerobic process
– Obligate & facultative anaerobes
– Less efficient in energy generation
Fermentation vs. Oxidation
• Used in biochemical testing based on:
– presence mixture of end products accumulates in medium
– Changes in pH
• Change in pH – KIA, TSI, CTA CH2O, HE
• Products of process: Methyl Red – mixed acids & VP - acetoin
• NOTE: if evaluating a CH2O & not glucose, no glucose should be in
the media
– Bacteria preferentially ferment glucose 1st
Lactose Fermentation
LF, NLF & sLF
• Key in ID of Enterobacteriaceae
• 1st Step
– ß-galactoside permease transports lactose across cell wall
• 2nd Step
– ß-galactosidase breaks galactoside bond releases glucose
• all organisms that ferment lactose ferment glucose
• sLF fermenters lack or have a weak β galactoside permease
– ONPG test to id sLF as does not require β galactoside permease
GPC
Staphylococcus spp.
•
•
•
•
(+) Catalase
Non-motile
Non-spore-forming
Most are facultative anaerobes
– •except S. saccharolyticus
•
•
•
•
S. aureus
S. epidermidis
S. saprophyticus
S. lugdunensis
– More virulent, may have mecA gene
Staphylococcus spp.
Isolation
• SBA
• Thioglycollate broth
• Selective media
□ Mannitol Salt agar
□ Columbia Colistin Nalidixic Acid agar (CNA)
□ Phenylethyl alcohol agar (PEA)
General characteristics
• GPC, in clusters
• Colonies - creamy, white, golden & may be “buttery-looking”
• Some β hemolytic
COAGULASE TESTS
SLIDE TEST
• Cell-bound coagulase (Clumping Factor)
• enzyme that clots human, rabbit, or pig plasma
• If (+) reported as Staphylococcus aureus
• If (-) the tube test must be done
TUBE TEST
• Free coagulase (staphylocoagulase)
• extracellular enzyme that causes clot to form when bacterial are incubated
in plasma
• If (+) reported as Staphylococcus aureus
– 5% of S. aureus do not produce cell-bound coagulase (CF)
• If (-) reported as CoNS
• Coagulase
– (+) Staphylococcus aureus
– (-) other staphylococci (CoNS)
– S. lugdunensis
• (+) Cellbound (CF) coagulase & (-) free (staphylocoagulase)
• Novobiocin on CoNS urine isolates
– (R) -Staphylococcus saprophyticus
– (S) other CoNS
• Hemolysis
– β: Staphylococcus aureus
– No hemolysis: S. epidermidis & S. saprophyticus
–
• Micrococcus spp.
– GPC, tetrads, irregular clusters
– (+) catalase
Micrococcus spp.
Oxidase
Staphylococcus spp.
(+)
(-)
Bacitracin
S
R
Lysostaphin
R
S
Coagulase
(-)
varies
• Staphylococcus aureus
– must perform antimicrobial susceptibility on all isolates
– Most penicillin(R) – beta lactamases
• MRSA, HA-MRSA, CA-MRSA
– Vancomycin VISA & VRSA
MRSA KEY TEST
• Oxacillin susceptibility but now Cefoxtin (better inducer of mecA-mediated
resistance) is preferred
• GOLD STANDARD
– Detection mecA gene – same day tests: Nucleic Acid probes or PCR
D TEST
• Macrolide resistance
• Inducible clindamycin resistance presence of erythromycin
Streptococcus spp.
• GPC, chains, pairs
• (-) catalase
• Most facultative anaerobes
• Streptococcus spp.
• Enterococci spp.
• LANCEFIELD CLASSIFICATION
– C carbohydrate on cell wall
– A, B, C, D, F, G & N – most common
Lancefield
Hemolysis
Term
S. pyogenes
A
ß, wide zone
Group A strep
S. agalactiae
B
ß – narrow zone
Group B strep
S.dysgalactiae, S. equi
C
ß
Group C strep
S. bovis group
D
α or none
nonenterococcus
Enterococcus faecalis &
E. faecium
D
α, ß or none
enterococcus
S. pneumoniae
No group
α
pneumococcus
α, ß or none
viridans strep
Anginosus group, mutans A, C, F, G, N, or -group, mitis group,
salivarius group
Susceptibility Tests
• Bacitracin
– identify Group A strep
– Low concentration 0.04 U selectively inhibits growth of Group A strep
• Sulfamethoxazole & Trimethoprim (SXT)
– used with bacitracin to improve accuracy Group A strep
– Groups A & B are resistant to SXT
• Optochin susceptibility
– used on α hemolytic strep
– ethylhydrocupreine hydrochloride
– Streptococcus pneumoniae is sensitive to optochin
• Bile Solubility (sodium deoxycholate & sodium taurocholate)
– Correlates well with optochin susceptibility
– (+) Streptococcus pneumoniae in solution lyses & solution clears
– (-) viridans streptococci do not lyse
• Hippurate hydrolysis
– Differentiate Group B strep from other ß hemolytic strep
– (+) Streptococcus agalactiae (GBS)
• PYR hydrolysis test
– Substrate is L-pyrrolidonyl-ß-naphthylamide & L-pyroglutamic acid-ß
naphthylamide
– (+) Streptococcus pyogenes & Enterococcus spp.
– (-) other streptococci
– more specific for Group A strep than bacitracin
– (+) Aerococcus spp. & Gemella spp
• CAMP test
– CAMP factor
– PRES ID Group B strep
– Enhanced hemolysis
• ß-hemolytic Staphylococcus aureus & CAMP
factor producing β- hemolytic or some nonhemolytic Group B strep
– (+) arrow-shaped hemolysis pattern
• Bile Esculin
– Grow in presence of 40% bile
– Hydrolyze esculin – black complex
– (+) Group D streptococcus & Enterococcus spp.
– (-) viridans Strep
• 6.5% sodium chloride broth
– (+) Enterococcus spp.
– (-) Group D
Enterococcus faecalis
• Leucine AminoPeptidase test (LAP)
(+) Streptococcus & Enterococcus spp. & Pediococcus spp.
– (-) Aerococcus spp. & Leuconostoc spp.
Susceptibility Testing
• Penicillin
– drug of choice for most streptococcal infections
– Penicillin resistant organisms
• Some Streptococcus pneumoniae & viridans streptococci
• Vancomycin
– Effective against GPC organisms
– Vancomycin resistance now being encountered
– Enterococci & some streptococci are showing resistance
Streptococcus pyogenes (Group A)
• Pharyngitis & tonsillitis
• Scarlet fever, skin infections, and other septic infections
• Rheumatic fever, acute glomerulonephritis
Laboratory
•
•
•
•
•
•
•
Pinpoint colonies, ß hemolysis
(+) PYR
(S) Bacitracin
(-) Hippurate hydrolysis
(-) CAMP
(-) Bile esculin
(-) 6.5% NaCl
Streptococcus agalactiae
• invasive disease in newborns
• Elderly, immunodeficient & women who become ill after childbirth or
abortion
• All pregnant women should be screened for GBS (group B Stept) at
35-37 weeks gestation
• Laboratory
– Grayish white colonies, small zone of ß hemolysis
– (-) catalase
– (+) Hippurate hydrolysis
– (+) CAMP test
PRES ID of ß hemolytic streptococci
ß-hemolytic colonies
Bacitracin
Susceptible
Resistant
Group A
Streptococcus pyogenes
CAMP
+
Group B
Streptococcus agalactiae
-
Possibly group D or ß hemolytic;
not group A, B
Group D streptococci
• Streptococcus bovis group
• group D antigen
• endocarditis, UTI, abscesses, wound infections & associated bacteremia
resulting from presence of gi tumors
• Penicillin
– (S) Group D
– (R) Enterococcus spp., usually
• Laboratory
– small, translucent colonies - α or no hemolysis
– (+) Bile Esculin
– (-) 6.5% NaCl
– (-) PYR
Enterococcus spp.
•
•
•
•
•
•
•
Normal flora in intestinal tract
Enterococcus faecalis – most common isolate
Enterococcus faecium
Group D antigen
UTIs followed by bacteremia & endocarditis
Show resistance to many antibiotics
VRE – Vancomycin Resistant Enterococcus
Laboratory
– Β or α or no hemolysis
– May have pseudocatalase reaction
– (+) Bile Esculin
– (+) 6.5% NaCl
– (+) PYR
PRES ID of Group D strep & Enterococcus
ß, α, or no hemolysis in SBA
Bile Esculin
-
+
Non-hemolytic streptococci
6.5% NaCl
+
Enterococcus spp.
PYR (+)
Group D streptococci
PYR (-)
Streptococcus pneumoniae
pneumococcus
• Normal RT flora
• Pneumonia, sinusitis, otitis media, bacteremia, meningitis & 30% acute
septicemia
• Quellung reaction – capsule swelling
• allows ID of Streptococcus pneumoniae & specific serotypes
• treat with penicillin but some strains show resistance & are treated with
erythromycin or chloramphenicol
• Vaccine – childhood & > 50 yrs.
Streptococcus pneumoniae
Laboratory diagnosis
– GPC - lancet shaped & usually have capsule
– SBA or Choc
– Some require increased CO2
– Large zone of α hemolysis
– round, glistening, wet, mucoid, dome shaped & with time collapse of
colony's center
– Optochin
• (S) S. pneumoniae
• (R) other α hemolytic streptococci
– Bile solubility
• (+) S. pneumoniae
• (-) other α hemolytic strep
viridans streptococci
•
flora URT, female genital tract & gi tract
• S. anginosus group, S.mutans group, S. mitis group, S. salivarius group & S.
bovis group
• Low Virulence, opportunist
• Most common cause of subacute bacterial endocarditis
• gingivitis, dental caries, meningitis, abscesses, osteomyelitis & empyema
• Penicillin but some resistant strains reported
Laboratory
– SBA may show zone of α hemolysis
– (-) Bile solubility
– (R) optochin
Abiotrophia spp. & Granulicatella spp.
• Nutritionally Variant Streptococci (NVS)
• Satellite colonies @ other bacteria
• Bacteremia, endocarditis, otitis media
• Should be suspected in blood cultures
Laboratory
– GPC resembling strep
– Do not grow on subculture
– Subculture
• Staphyloccous aureus streak
– Satellitism
GNC
Neisseria spp.
• Aerobic, non-motile, non-spore-forming
• GNDC
• (+) Oxidase
• (+) Catalase
– Except for:
• N. elongata GNR (-) catalase
• N. weaveri GNR (+)catalase
• Capnophilic
• fastidious organisms, need enriched media & moisture for optimal recovery
• Neisseria gonorrhoeae (true pathogen); N. meningitidis (commensal URT
of carriers) & other Neisseria spp. (opportunistic pathogens)
Neisseria gonorrhoeae
• urogenital tract infections & conjunctiva
• Disseminated infections – joints, meningitis
• Specimen: males–urethra; females–endocervix & rectum, pharynx &
joint fluid
• Best chance for recovery is direct plating of specimen to gonococcalselective media
• incubate at 35O C in 3-5% CO2
• PCR assay N. gonorrhoeae vs. Chlamydia spp.
Neisseria gonorrhoeae
Direct Microscopic exam
• urogenital specimens
• Not recommended for pharyngeal specimens
• Evidence of gonococcal infection
– GNDC intracellular from symptomatic male with discharge
correlates at a rate of 95% with culture
– Females have vaginal commensal flora that resemble gonococci
– culture is necessary
• Gram stain >5 PMNs/field but no bacteria may suggest NonGonococcal Urethritis (NGU)
• •NGU rule out: Chlamydia trachomatis & Ureaplasma urealyticum
Neisseria gonorrhoeae
• Requires chocolate agar (+) selective agents
• Thayer-Martin (TM)
• Vancomycin (GP) , Colistin (GN), Nystatin(fungi)
• •Modified Thayer-Martin (MTM)
– Vancomycin, Colistin, Nystatin, Trimethoprim (Proteus spp.)
• Martin Lewis (ML)
– Vancomycin,Colistin, Anisomycin (fungi), Trimethoprim
• New York City (NYC)
– Vancomycin, Colistin, Amphotericin B (fungi), Trimethoprim
Neisseria gonorrhoeae
PRES ID
1. Small, gray/tan, transluscent raised colonies 24-48 hrs.
2. Gram stain to verify GNDC kidney bean-shaped
–
GNRs Kingella spp. & Acinetobacter spp. may grow on gonococcal media ,may
look similar, to differentiate, inoculated onto plate with 10 unit penicillin disc,
growth on edge of zone of incubation to evaluate morphology
3. (+) Oxidase
– Must be done on all isolates
Antimicrobial resistance
– All strains must be tested for ß-lactamase production
– Must be performed on primary culture plate as plasmid can be lost on
subculture
Neisseria gonorrhoeae
Definitive Identification
• Other oxidase (+) GNDC, can be recovered from gonococcal selective
media - Neisseria cinerea, N. meningitidis, Moraxella catarrhalis
CH2O Utilization
• Cystine Trypticase Agar (CTA)
– (+) Glucose fermentation & (-) Maltose, sucrose & lactose
fermentation
Chromogenic enzymes
• Multitest
• Matrix assisted Laser Desorption/Ionization Time of Flight (MALDI Tof)
Non-culture
• Nucleic Acid Amplification Test (NAATs)
Neisseria meningitidis
meningococcus
• meningitis, meningococcemia, rare pneumonia, purulent arthritis & endophthalmitis
• nasopharynx & oropharynx of 3-30% of asymptomatic individuals
Laboratory
• CSF, blood, nasopharyngeal swabs & aspirates, sputum & urogenital sites
• Direct exam
– intracellular & extracellular GNDC & may have capsule
– Helps to concentrate to specimen
• grows on gonococcal-selective agars, SBA & Choc
• small, gray, sometimes mucoid, convex colonies
• (+) Oxidase
• (+) Catalase
• CTA sugar:s (+) glucose & maltose fermentation & (-) sucrose & lactose
Moraxella catarrhalis
• GNDC
• Normal commensal RT, opportunistic pathogen
• Pneumonia, sinusitis, otitis media & systemic disease
Laboratory
• Grows on SBA & Chocolate
• smooth, opaque, gray to white colonies - “hockey puck”
• Usually inhibited by colistin in gonococcal selective media
• (+) Oxidase
• (+) Catalase
• Asaccharolytic in CH2O degradation tests •
• (+) DNase
• (+) Butyrate esterase
Fastidious GNR
• HACEK
•
•
•
•
•
•
•
•
•
•
•
Haemophilus spp.
Aggregatibacter spp. (formerly Actinobacillus)
Cardiobacterium hominis
Eikenella corrodens
Kingella spp.
Capnocytophaga spp.
Pasteurella spp.
Brucella spp.
Francisella spp.
Legionella spp.
Bordetella spp.
HACEK Group
Characteristics
– GNR/CB
– Capnophilic
– Important cause of endocarditis
• flora of oral cavity – allows for introduction into blood stream
Haemophilus spp.
• GNCB, pleomorphic
• Most nonpathogenic or opportunistic pathogens
• X factor-hemin & V factor (NAD) “para” only V factor
• H. influenzae
H. parainfluenzae H. haemolyticus
• H. parahaemolyticus
H. aphrophilus
H. paraphrophilus
• H. aegyptius
H. ducreyi
•
Haemophilus spp.
• Hemolysis on 5% horse’s or rabbit’s blood to differentiate
• SBA only X factor available
– V factor dependent do not grow on SBA
• Chocolate agar
– Lysed rbcs by heat results in containing both X & V factors
• Satellitism
– Staphylococcus aureus, Streptococcus pneumoniae, Neisseria spp.
produce V factor as a byproduct of metabolism
• Haemophilus influenzae
– Meningitis – decrease frequency due to vaccine (Hib)
– Epiglottitis
• •Haemophilus aegyptius - acute contagious conjunctivitis
“pinkeye”
• •H. influenzae biogroup aegyptius -conjunctivitis
• •H. ducreyi - Chancroid – painful, soft
– highly communicable STI Genital Ulcer Disease (GUD)
• Laboratory
– Almost any specimen but commonly blood, CSF, middle-ear
exudates, joint fluids, URT & LRT specimens, swabs conjunctival
swabs, vaginal swabs, abscess drainage
– Prompt transportation & processing is critical
Haemophilus spp.
• Culture
– Does not grow on SBA, grows on Chocolate
• H. ducreyi
– Special media, hold for at least 7 days at 33O C
• H. aegyptius
– Special media, hold for at least 4 days
• Colony morphology
– On chocolate translucent, tannish, moist, smooth, convex colonies
with “mousy” or “bleach-like” odor
• Microscopic morphology on direct smear
– GNCB to long filaments
– H. ducreyi “school of fish” formation
– H. influenzae – intra & extracellular
Haemophilus spp.
• 1st clue to a Haemophilus spp.
– No Growth (NG) on SBA, MAC or other enteric agars
– GNCB, pleomorphic on Chocolate agar
• X & V factor test
• Porphyrin test
– (+) Porphyrin heme producing will fluoresce
– (-) Porphyrin non-heme producing will not fluoresce
• Many strains produce ß-lactamase – rapid tests
– Chromogenic cephalosporin test
– Acidometric test
– If (+) strain is resistant to ampicillin & amoxicillin
• Aggregatibacter aphrophilus
– X-dependent & X-independent strains
– (-) catalase
– granular, yellow with center opaque zone colonies on Choc
• Aggregatibacter actinomycetemcomitans
o (+) catalase
o BA “star” 4-6 points in center 100x
• Cardiobacterium hominis
– GNR or GV – forms rosettes
– Slow grow SBA & CHOC – may see pitting
Eikenella corrodens
•
•
•
•
•
•
•
flora mouth & gi
Usually polymicrobial infection, often trauma - human fights or bites
GNCB
(-) motility
(+) oxidase
(-) Catalase
asaccharolytic
• often produce yellow pigmented colonies
– @45% pit the agar (if see 1st think Eikenella spp.)
– Bleach-like odor
• NG on Mac or EMB
o Kingella spp.
o Often follow viral infection or poor oral care or oral surgery
o GN CB/short rods with squared ends, in pairs or short chains, tend
resists decolorization
o Nutritionally fastidious
o “twitching” motility (no flagella)
o Capnocytophaga Spp.
–
–
–
–
–
–
–
o
Resemble HACEK
Normal flora mouth & oral cavities
GNR - Thin & fusiform
Fastidious, pitting but fermentative
May see gliding motility on agar surfaces
Often yellow-orange pigment
Isolated from blood from granulocytopenic patients who have oral
ulcers, juvenile periodontal disease, and endocarditis
Pasteurella multocida
•
•
•
•
Most common isolated Pasteurella spp.
Zoonosis
GNCB, pleomorphic - Bipolar staining
Laboratory
– SBA nonhemolytic
– (+) oxidase
– (+) catalase
– weak TSI (glucose fermentation weak acid production)
– (+) ODC
– (+) Indole
– (+) Urease
– Does not grow on Mac
Brucella spp.
•
•
•
•
Zoonosis
Brucellosis, Undulant fever, Mediterranean, Crimean & Malta Fevers
aerosol, percutaneous & oral routes of exposure
remittent fevers, fluctuate regularly at consistent intervals & persist for
days, months, or years
• B. melitensis most common cause of brucellosis
• Category B Select Biological Agent
– Easily disseminated & moderate morbidity but low mortality
– BSL 3 lab – lab exposure results in 30%-100% acquiring infection
• Blood & bone marrow cultures performed with acute & convalescent
sera for serologic testing
Brucella spp.
Laboratory
• Tiny, GNR/CB
• facultative intracellular pathogen in phagocytes
• Grow aerobically on Choc & BA in 18 hrs - hold 4 days (also TM or Martin
Lewis)
• Smooth, raised & translucent colonies
• 50% grow on MAC
• (-) motility
• (+)oxidase
• (+) catalase
• (+) Nitrate reduction
• (+) urease
Francisella spp.
• Zoonosis –tularemia or “rabbit fever”
• ingestion, inhalation, arthropod bite or contact infected tissues
• Category A agent - easily disseminated (ID50 = 50), high mortality rates
• BSL 3
Laboratory diagnosis
• tiny, poor staining GNCB
• NG on Mac & may initially grow on SBA
• CHOC, MTM, BCYE with cysteine for 36-48 hours
• Smooth, gray-white, raised colonies
• (+) catalase - weak
• (-) Oxidase
• (-) urease
• (+) beta-lactamase test
• (-) X & V factors
Legionella pneumonophila
• Weakly staining tiny GNR, pleomorphic
• Intracellular (phagocytes) & extracellular
• Legionnaires’ disease & Pontiac fever
Laboratory
• Suspect if NG on SBA
• Acid wash sputum & plate on selective media for best isolation
• Subculture on BYCE & BYCE with L-cysteine
– 3-5 days “ground-glass” center of gray & granular with
pink/light blue periphery of colony
– As age - gray/white or blue/green, convex, glistening colonies
• Urine for antigen detection
– Detects L. pneumophilia serogroup 1 soluble antigen
Bordetella pertussis
• Whooping cough
• One most highly communicable diseases of childhood
• DPT vaccine
Laboratory
• Tiny GNR or GNCB
• Slow growing 3-5 days
• Regan-Lowe Charcoal media
– Young colonies - mercury droplets, as age turn whitish gray
• Bordet-Gengou Potato Infusion agar
– Small, white, domed “pearls”
– B. pertussis & B. parapertussis – hemolytic
• Nucleic acid detection by PCR
– Primary rapid diagnostic tool
– Nasopharyngeal swabs
ENTEROBACTERIACEAE
ENTEROBACTERIACEAE culture
• SBA or Choc - Large, grayish, smooth colonies, some
hemolytic
• MacConkey (MAC) - lactose in media
– LF -pink colonies & NLF –tan/colorless colonies
• Eosin-Methylene Blue (EMB) lactose & sucrose in media
– LF- Pink colonies
– E. coli blue-black/green sheen colonies
– NLF & NSF -translucent colonies
STOOL CULTURES
Enhance Salmonella spp. & Shigella spp. growth
HE Agar lactose & sucrose in media
• LF &/or SF–yellow colonies
• NLF & NSF –green colonies
• H2S – black centers
XLD Xylose-Lysine Deoxycholate (lactose, sucrose & xylose)
• LF &/or SF &/or XF–yellow colonies
• NLF & NSF & NXF - red colonies
• H2S – black centers
• Salmonella spp. (+) lysine decarboxylase
Enterobacteriaceae Identification
• (-) oxidase except Plesiomonas shigelloides
• (+) glucose fermentation
• (+) NO3 reduction
• (+) motile except Shigella spp. , Klebsiella spp. &
*Yersinia spp.
*Yersinia spp. (+) motile at 250 C
KIA or TSI PRES ID of Enterics
• KIA = lactose in slant & glucose & H2S in butt & +/- gas
• TSIA= lactose & sucrose in slant; glucose & H2S in butt & +/- gas
• Phenol red indicator
– alkaline pH= red & acid pH=yellow
• Slant=aerobic & butt=anaerobic
NOTE:
• Enterobacteriaceae if A/A or K/A
• Not an Enterobacteriaceae if K/K or A/K
TSIA & KIA Interpretation
• ONPG
o-Nitrophenyl-p-D-galactopyranoside
– Determines if slow LF
• CH2O fermentation
– ability to ferment a specific 1% concentration of carbohydrate
• MRVP
– Methyl red (MR)
• Mixed acid fermentation pathway (strong acids)
– Vogues-Proskauer (VP)
• Butylene glycol pathway byproduct acetoin (acetylmethylcarbinol)
• (+) Klebsiella spp., Enterobacter spp. Serratia spp. & Hafnia sp. group
• DecarboxylaseTests
– Decarboxylase remove carboxyl group of an amino acid
– Lysine, ornithine & arginine
• PAD Phenylalanine Deaminase
– deaminates phenylalanine to phenylpyruvic acid
– (+) PPM group (Proteus, Providencia & Morganella)
• Citrate Utilization Test
– Utilize sodium citrate as a sole carbon source
• DNase
– Extracellular DNase produced by some bacteria
– (+) most Serratia spp.
• Gelatin Liquefaction
– Proteolytic enzymes break down gelatin into amino acids
• Indole
– Kovacs or Ehrlich
– Degradation products of amino acid tryptophan
– Add paradimethylaminobenzaldehyde -red color
• SIM
– H2S production
– Indole production
– Motility
• Malonate
– use sodium malonate as a sole carbon source
• Motility
• Nitrate reduction - NO3 to NO2 or to N (gas)
– 1st Step - add sulfanilic acid & N,N-dimethy-α-naphthylamine
• (+) Red
– If no color change go to 2nd Step - add zinc
» (-) Red and (+) No color change
• •Cytochrome oxidase
– Oxidizes reduced cytochrome with molecular oxygen
• Lysine Iron agar (Slant/Butt)
– Purple/purple = (+) Lysine decarboxylase
– Purple/yellow butt = (-) Lysine decarboxylase
– Cherry red/ yellow butt = (+) Lysine deaminase
– H2S production masks (+) lysine decarboxylase reaction
• Urease
– Breaks down urea to NH3 which decreases pH
Enteric Pathogens
• Salmonella, Shigella, Aeromonas, Campylobacter, Yersinia, Vibrio and E. coli
0157:H7, Plesiomonas shigelloides
• •All fecal specimens should be routinely screened for
– Salmonella, Shigella, and Campylobacter
– Many labs routinely screen for E. coli 0157:H7
• Serologic groupings for Salmonella spp. & Shigella spp.
– Based on O antigens
– Slide Test
• 1 drop antisera onto slide & add saline emulsion containing isolate. If
(-) may be due to capsule & requires emulsion heated to 1000 C to
remove capsule & then re-test
Escherichia coli
• MAC – LF, bile precipitation
• SBA – may appear ß-hemolytic
• EMB – green metallic sheen
• IMViC + + - • TSI= A/A (+) gas
• (-) for DNAse; urease & PAD
LF
Escherichia coli
•
•
•
•
•
Most common cause UTIs
Meningeal (especially newborn)
Gastrointestinal infections
Wound
Bacteremia
• Five major categories of diarrheogenic E. coli
– Enteropathogenic (EPEC)
– Enterotoxigenic (ETEC)
– Enteroinvasive (EIEC)
– Enteroadherent
– Enterohemorrhagic (EHEC) serotype 0157:H7
• produces Verotoxins I and II
LF
Enterohemorrhagic E. coli (EHEC)
0157:H7 strain
• SMAC
– MAC with sorbitol instead of lactose (SMAC)
– E. coli 0157:H7 does not ferment sorbitol
• MUG
– Screen for E. coli 0157:H7
• Rarely produces Beta -glucuronidse
• 92% of other strains do produce it
• SMAC (-) &/or (-) MUG
– Serotype to confirm E. coli 0157:H7 with anti-serum
KLEBSIELLA TRIBE
LF
• Klebsiella spp., Enterobacter spp., Serratia spp., Pantoea spp.,
Cronobacter spp. & Hafnia spp.
•
•
•
•
•
•
(-) H2S
(+) urease few
(-) MR
(+) VP
(+) Citrate most
(-) indole most
KES – most IMViC
- - + + except K. oxytoca +-++
Klebsiella spp. LF
• K. pneumoniae
– Nosocomial
– Frequent cause of LRT infections
– wound infections, UTI, bacteremia
• K. oxytoca
– infections similar to K. pneumoniae
• IMViC - - + + except K. oxytoca indole (+)
•
•
•
•
TSIA = A/A, no H2S, no gas
(-) motility
(+) urea
(-) ornithine decarboxylase
Enterobacter spp. LF
•
•
•
•
•
•
•
Enterobacter aerogenes
E. cloacae
Isolated from wounds, urine, blood & CSF
Mucoid colonies
(+) motility
IMViC - - + +
TSI A/A, no H2S, no gas
• E. aerogenes = (-) urease & (-) arginine decarboxylase
• E. cloacae = (+) urease (65%) & (+) arginine decarboxylase
Serratia spp.
SLF
• Serratia marcescens
• S. liquefaciens
• S. rubidaea
• S. marcescens
– Opportunistic pathogen
– nosocomial outbreaks – UT ; RT, bacteremia-nurseries, cardiac surgery &
burn units
• Resistant to a wide range of antimicrobials
– Susceptibility test MUST be performed on every isolate
– some pink pigment at 300 C & look like a LF MAC at 18 hr even though
SLF
Serratia spp.
sLF
Serratia marcescens
• TSI = A/A (Ferments sucrose)
• KIA – K/A in the 1st 18-24 hrs & after that A/A
• (+) ONPG to id SLF
• IMViC - - + +
• (+) DNase
• (+) Motility
Proteus spp., Morganella spp. & Providencia spp. NLF
• Normal intestinal flora - Opportunistic pathogens
• Differentiate from other Enterobacteriaceae
– (+) PAD
– (+) Lysine deaminase
• Proteus spp.
–
–
–
–
–
–
Proteus mirabilis
Proteus vulgaris
urine, wounds, ear s & bacteremia
swarming colonies on non-selective media -SBA
“burned chocolate” odor
(+) H2S production
PPM Group
• Morganella morganii
– May UTI & diarrhea
• Providencia spp.
– P. rettgeri
• UTIs & caused occasional nosocomial outbreaks
– P. stuartii
• nosocomial outbreaks burn units & been isolated from urine
• cannot differentiate between Morganella & Providencia spp. with
standard tests
P. mirabilis P. vulgaris
Morganella
morganii
Providenica
rettgeri
Indole
-
+
+
+
Methyl Red
+
+
+
+
VP
+ (50%)
-
-
-
Citrate
+ 65%)
-
-
+
Motility
+
+
+
+
Urea
+
+
+
+
Alk/Acid
Acid/Acid
Alk/Acid
Alk/Acid
TSI
Ferment sucrose
KIA
Alk/Acid
Alk/Acid
Alk/Acid
Alk/Acid
H 2S
+
+
-
-
PAD
+
+
+
+
Edwardsiella tarda NLF
•
•
•
•
•
•
Bacteremia & wound infections
(-) Urea
(+) Lysine decarboxylase
(+) H2S
(+) Indole
(-) Citrate
Citrobacter spp. LF
•
•
•
•
(+) urea slowly
(+) citrate
(+) methyl red
C. freundii
– UTI, pneumonias, intra-abdominal abscesses
– (+) H2S & some do not ferment lactose & can resemble Salmonella
when isolated from stools
Salmonella spp.
• Typhoid fever
– Most severe form of enteric fever is caused by S. serotype Typhi
– Other enteric fevers - S. serotype Paratyphi, S. serotype Choleraesuis
• Non-typhoidal bacteremia
• Carrier state
• MAC, HE & XLD
•
•
•
•
•
IMViC - + - +
TSI = K/A, no gas, (+) H2S
(-) PAD
(-) Urease
(+) lysine decarboxylase
Shigella spp. NLF
• True Pathogen that causes bacillary dysentery
– S. dysenteriae (group A)
– S. flexneri (group B)
– S. boydii (group C)
– S. sonnei (group D) – most common isolate US
• S. dysenteriae
– blood, mucous & pus in stool
• Media
– MAC, HE & XLD
– SS agar - inhibits GP, most coliforms & swarming Proteus spp.
• LF (pink) from NLF & H2S production
Shigella spp. NLF
•
•
•
•
•
(-) motile
(+) glucose fermentation without gas except some S. flexneri (+) gas
(-) Urea
(-) H2S
(-) lysine decarboxylase
•
•S. sonnei
– (+) ornithine decarboxylase
– SLF after 48 hrs & see pink colonies on Mac
– (+) ONPG
Yersinia spp.
NLF
• Y. pestis – plague
–
–
–
–
–
–
GNR short, plump
Wayson Stain - bipolar staining “safety-pin”
Grows at 37OC but likes 25-30O C best
Class A Bioterrorism Agent
(-) urease
(-) motility at 250 C & 350 C
• Y. pseudotuberculosis - Similar to plague
– Pseudotubercles (caseous swellings)
– (+) motility at 25OC & (-) at 35O C
– (+) urease
Yersinia enterocolitica NLF
 acute enteritis; appendicitis-like syndrome, arthritis & erythema nodosum
• contaminated meat, esp. pork (chitterlings), vacuum-packed deli meet,
• Optimal growth 25 – 30O C
• (+) motility at 25OC & (-) at 35O C
• TSI A/A (-) H2S ferments sucrose
• •CIN Selective Media
• cefsulodin, irgasan, novobiocin, bile salts & CV inhibitory
• Inhibits normal colon organisms better than Mac
• Yersinia-selective agar (YSA) base- add mannitol to make differential
– (+) Mannitol fermentation colony has red center