identification of pathogenic bacteria by laboratory methods m. kent froberg, md

Identification of Pathogenic Bacteria by Laboratory Methods

M. Kent Froberg, MD

Laboratory Methods

• ID of pathogenic bacteria using series of differential tests

differential & selective media morphology & staining biochemical properties antibiotic resistance or susceptibility plasmid, ribosomal or DNA analysis

Gram Positive Cocci

• Staphylococci vs Streptococci – Staph aureus catalase +, coagulase +,

& -hemolytic – Strep , , or non-hemolytic, catalase -

Catalase

• Enzyme that converts H2O2H2O and O2

• Visible bubbles when drop of 3% H2O2 is added to culture of catalase + bacteria

Catalse + Catalse -

Catalase - Catalase +

Between Staph Species

• S aureus coagulase + (clots plasma)

• S epidermidis & saprophyticus coagulase neg– S saprophyticus novobiocin resistant– S epidermidis novobiocin sensitive

Lower tube shows fibrin clot following addition of plasma to culture of Stap aureus. Top tube is negative indicating non-pathogenic Staph.

Left: novobiocin resistant Staph saprophyticus

Right: novobiocin senstitive Staph epidermidis

Between Strep Species

• Hemolysis:– Strep pyogenes & agalactiae hemolytic – Enterococcus variable hemolysis – Strep viridans & pneumoniae hemolytic

Beta-hemolysis with clearing of blood agar

Green discloloration of alpha-hemolysis

No hemolysis on blood agar

More Strep Tests

• Strep pyogenes bacitracin sensitive

• Strep agalactiae & Enterococcus bacitracin resistant

• Strep viridans resistant to optochin

• Strep pneumoniae sensitive to optochin

Zone of inhibition around optochin disk - presumptive identification of Strep pneumoniae

Motility Tests

• Many bacteria show motility in media • Agar tube inoculated with needle stab • Media contains tetrazolium bacteria

reduces to formazan (red color) • Location of bacteria determined by location

of red color• Useful for Borrelia, Vibrio, Salmonella, E

coli, etc.

2,3,5-Triphenyltetrazolium chloride (TTC) Formazan (red color)

Left tube: motile Center: nonmotile Right: control

Oxidase Tests

• Presence of cytochrome oxidase

• Neisseria & Pseudomonas +

• Enterobacteriaceae neg

• Test converts colorless tetramethyl-p-phenylenediamine to oxidized form (deep purple/blue)

Oxidase + colony at 12 o’clock, 3 & 9 o’clock cultures are negative

Oxidase test done on paper saturated with oxidase reagent. Pseudomonas on left (+), Staph aureus on right (-).

Enterobacteriaceae

• API 20 E series of substrates and indicators that are inoculated with unknown and media

• Tests for enzymes and carbohydrate utilization

• Pattern of + and - results compared to a table of known results to identify unknown

cultureno.

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

identification

8101 + – + + – – – – + – – + + – + + + + – +Escherichia

coli

5B + – – – + – – – – + – + + + + + – – + +Enterobacteragglomerans

8P44 – – + + – + – – + – – + + – – – + – – +Edwardsiellahoshinae

Oxidation-Fermentation Tests

• Mostly used to distinguish fermentative Enterobacteriaceae from oxidative Pseudomonas & Bordetella

• Grow isolate in low agar content media in presence of different sugar substrates and pH indicator

• Stab media: one tube sealed with mineral oil to promote anaerobic growth/ other tube unsealed

• Indicator yellow at pH 6.0/ green at pH 7.1, blue at pH 7.6

Mineral oil covers media in tubes 1 & 3. Pseudomonas in tubes 1 & 2, Shigella in tubes 3 & 4.

Enterobacteriaceae

• Enterotube II 12 compartments of various media allows determination of 15 different characteristics of organism

• Positive results given a number, added in groups and sums combined to create unique 5-digit identifier in computer databank

Question #1

1. The laboratory obtains a surgical wound swab from a 56 year old male four days after undergoing a colectomy for colon cancer. The isolate is gram +, catalase +, coagulase + and hemolytic. It is most likely

a. Streptococcus pyogenes b. Enterococcus faecalis c. Staphylococcus aureus d. Staphylococcus saphrophyticuse. Streptococcus viridans

Question #2

2. The initial data necessary to adequately identify a bacterial unknown are its

a. Ribosomal sequence

b. Antibiotic resistance pattern

c. Motility

d. Ability to ferment glucose

e. Morphology and gram staining

Question #3

3. Streptococcus viridans and pneumoniae are both hemolytic, catalase -, gram + cocci. Which test may readily distinguish them?

a. Enterotube II

b. Novobiocin resistance

c. Motility test

d. Oxidase test

e. Resistance to optochin