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Bacterial Identification The Essentials for Plate Round

Rongpong Plongla, MD. MSc.

Oct 07, 2011

www.idchula.org

Diagnosis of Bacterial Infection

culture

on plates or in broth

identification by biochemical or

serological tests on pure growth from

single colony

microscopy

Decolorise Counterstain Stain

unstained or stained with e.g. Gram stain

sensitivities

Serodiagnosis DNA technologies

by disc diffusion

methods,

breakpoints or

MICs

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Identification of Bacteria

• Morphology

• Growth requirements

• Biochemistry

• Enzymes

• Antigens

Always Starting with Gram Staining and 3 Plates

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Gram-Positive Cocci

• 1. differentiate Staphylococci VS Streptococci – Using Gram stain morphology

• Staphylococci: in cluster

• Streptococci: in pair or in chain

• *Micrococci: in quadruplets/Tetrads with lemon-yellow colonies

– Catalase test • Staphylococci: positive

• Streptococci: negative • This enzyme is responsible for protecting bacteria from hydrogen peroxide (H2O2) accumulation,

which can occur during aerobic metabolism. • Catalase breaks H2O2 down into water and O2 • * most aerobic organism make catalase i.e Staphylococci, Micrococci, Rothia, Pseudomonas

aeruginosa, Serratia marcescens, Aspergillus, Nocardia, Burkhodelria cepacea

Staphylococcus

• 1. looking at Coagulase test to distinguish S. aureus

– Positive: S. aureus • S. aureus colonies is usually yellow.

– Negative: Coagulase-Negative Staphylococci

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Staphylococcus

• 2. distinguish S.saprophyticus from other Coagulase-negative Staphylococci (i.e. S. epidermidis) with Novobiocin Susceptibility

– S. saprophyticus: resistant to novobiocin

– S. epidermidis: sensitive to novobiocin

S. saprophyticus caused UTI

Streptococci

• 1. looking at hemolysis on horse/sheep blood agar, then grouping into beta vs non-beta hemolysis

Beta: complete hemolysis- transparent Alpha: partial hemolysis-greenish Gamma: no hemolysis

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Beta-hemolysis Streptococci

• 1. Streptococcus Group A (S.pyogenes)

• 2. Streptococcus Group B (S.agalactiae)

• 3. Streptococcus Group C, F, G

B eta-hemolytic Streptococci

• 1. looking at A-disc (Bacitracin disc) for Streptococcus pyogenes (group A): GAS

– Streptococcus group A : sensitive to bacitracin

– Also PYR positive

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Beta-hemolytic Streptococci

• 2. if A-disc is negative, identify Group B Streptococcus using 2 methods

– 1. CAMP test

– 2. Hippurate Hydrolysis + -

Christie-Atkins, Munch-Petersen (CAMP) test

• Detects the production of

enhanced hemolysis that occurs

when b-lysin and the

hemolysins of Group B

streptococci come in contact on

sheep blood agar

• Organisms that positive CAMP test

– GBS

– Listeria ivanovii

– Rhodococcus equi

Group B streptococci showing the

classical “arrow-shaped hemolysis

near the staphylococcus streak

+ -

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Reverse CAMP test

Positive and negative results. GBS is streaked down the center of the

plate the test organisms, Clostridium

sp., are streaked at right angles to the GBS. • Positive reaction of C. perfringens: “bow-tie" zone of hemolysis. •Alpha toxin + CAMP factor • Negative reaction of C. septicum: shows no hemolysis.

Beta-hemolytic Streptococci

• If A-disc, Hippurate test and CAMP test are negative

Streptococcus Group C, F, G

If you want to know the species (which almost not necessary): anti-sera or API

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Non-beta hemolytic Streptococci

• Streptococcus pneumoniae • Group D Streptococci

– Enterococci – S. bovis

• Viridans Streptococci – anginosus group (formerly milleri group: small

colonies and small, round shape gram positive bacteria )

– sanguinis group – mitis group – mutans group

Non-beta hemolytic Streptococci

• 1. using P-disc (Optochin disc) to identify S.pneumoniae

A zone of inhibition of 14 mm. or more in diameter will be seen around the disk after

incubation if the organism is Streptococcus pneumoniae. Other alpha-hemolytic streptococci are resistant to (not killed by) optochin.

If it’s S.pneumoniae There’re the MICs issues about whether meningeal or non-meningeal isolates

**note that 1. Lancet shaped diplococci with capsule are quite unique for S. pneumoniae 2. S.pneumoniae colonies can be mucoid, flat, and umbilicated

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• 2. if P-disc shows negative result, the next step will be using Bile esculin test (esculin hydrolysis) to demonstrate Group D Streptococci ( Enterococci or S. bovis)

• 3.if Bile esculin test is negative – It’s Viridans Streptococci API

• If Bile esculin test is positive, you must distinguish Enterococci vs S.bovis by using – Ability to growth in 6.5% NaCl

• Enterococci : positive

• S.bovis: negative

– PYR test • Enterococci : positive

• S.bovis : negative

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PYR test

• Organisms possessing the enzyme L-pyrrolidonyl arylamidase can hydrolyze the disk substrate L-pyrrolidonyl-ß-naphthylamide to release L-pyrrolidone and ß-naphthylamide.

• A positive reaction is a yellow to red color change. – S. pyogenes (group A)

– Enterococci

• 4. Species identification for group D non-enterococci (S. bovis) API (with confused nomenclatures)

• But, for enterococciusing tellurite agar

– E. faecalis (growth on tellurite medium)

– E. faecium

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• 5. if there’re gram-positive organisms arranged in chain grow on H/C bottle, but they’re not grow on Blood agar plate

– Nutritionally variant Streptococci

• Abiotrophia defectiva

• Granulicatella spp.

• Gemella spp.

– Required pyridoxine and cysteine for growth

• Human blood agar or staphylococcal streak tecnique

Gram positive cocci

Staphylococcus

Coagulase

+ -

S. aureus Staphylococcus

Coagulase-negative

Streptococci

Hemolysis on sheep blood

β α γ

Bacitracin (A-disc) PYR*

Optochin (P-disc)

Strep gr.A

Hippurate

Strep gr.B Other gr. C,G,F

S.pneumoniae Bile esculin

Strep not gr. D (γ) Viridan Strep (α)

6.5% NaCl, PYR

S. bovis Enterococci

+ - Catalase

S R

+ -

S R

+ -

+ -

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Gram-Negative Organisms

• 1. Guessing from Gram Stain – Large gram-negative bacilli

• Enterobacteriaceae, Glucose fermenters

– Slender-shaped small gram-negative bacilli • Non-glucose fermenters/ glucose non-fermenters

– Curved gram-negative bacilli • Vibrio spp., • Micro-aerophilic: Campylobacter spp., Helicobacter spp.,

Arcobacter spp.

– Coccobacilli, Diplococci • Haemophilus spp., Morexella spp, Neisseria spp, Acinetobacter

spp., Kingella spp., • Veillonella spp.(anaerobe)

• Safety pin/Bipolar staining??? – Provide no clues

• Burkholderia pseudomallei

• Pseudomonas aeruginosa

• Klebseilla pneumoniae

• Yersinia pestis

• Pasturella multocida

• Klebsiella granulomatis [Previously: Calymmatobacterium granulomatis] (cause of donovanosis)

• Francisella tularensis

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• 2. looking at MacConkey Agar

– If colonies are PINK : Lactose-fermenting organisms

• E.coli, Klebseilla, Serratia, Enterobacter, Citrobacter etc

• If organisms grow the colorless colonies

– Lactose-nonfermenting organisms

• Non-glucose fermentors

• Ferment glucose, but not ferment lactose

???????????

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• 3. TSI will help

– ? Or make more confusion :D

– WTF is TSI?

TSI

• TSI is composed of – 0.1% of glucose – 1% of lactose – 1% of sucrose

• Plus phenol red as the indicator – below 6.8 it is yellow – above 8.2, it is red

• If only glucose is fermented, acid produced in the butt will turn it yellow, but insufficient acid products are formed to affect the methyl red in the slant.

• However, if either sucrose or lactose are fermented, sufficient fermentation products will be formed to turn both the butt and the slant yellow.

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• เม่ือ Inoculate เชือ้ลงไปเชือ้ท่ี slant จะมีปริมาณมากกว่าบริเวณอ่ืน bacteria จะใช้น า้ตาล glucose หมดเร็วกว่า ถ้าเชือ้ไม่สามารถใช้ lactose และ sucose ได้จะหนัไปใช้ protein(polypeptone) ท่ีมีอยู่ใน media แทน ท าให้ slant มีภาวะ เป็นดา่ง เปล่ียน Phenal red เป็นสีแดง สว่น bott นัน้มีแบคทีเรียที่เจริญอยู่น้อยกว่า slant ภายใน 24 ชัว่โมงจะยงัใช้ glucose ไม่หมดจงึมีความเป็นกรดอยู่ (เปล่ียนสี phenal red เป็นสีเหลือง) ปฏิกิริยาจงึเป็น K/A ( Slant/Butt)

• ถ้าเชือ้สามารถใช้น า้ตาลได้ตัง้แต ่2 ชนิดขึน้ไป จะท าให้อาหารทัง้หลอดเป็นสีเหลอืง ปฏิกิริยาจงึเป็น A/A

• ถ้าเชือ้ไม่สามารถใช้น า้ตาลทัง้ 3 ชนิดได้เลย ปฏิกิริยาอาจเป็น N/N , K/N , A/N ( N คือไม่มีการเปล่ียนแปลง )

• ถ้าเชือ้ใช้น า้ตาลแล้วให้ gas แล้ว gas จะดนัให้ media ยกตวัขึน้ หรือมีรอยแยกของ media • ถ้าเชือ้สร้าง H2S จะท าปฏิกิริยากบั Ferrous ion ท่ีมีอยู่ใน mediaได้ ferous sulfide ท า

ให้เกิดเป็นตะกอนสีด าขึน้

• When you see TSI you should mention – 1. Butt

– 2. Slant

– 3. Gas

– 4. H2S

Yellow=acid

Pink=alkali

ก้นเหลือง=ferment glucose

เหลืองทัง้หลอด=ferment ทัง้ glucose+lactose+/-sucrose

ไม่เหลืองเลย=glucose non-fermentors

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Glucose fermentation

+ -

Oxidase

+ -

Lactose ferment

+ -

OF glucose (OXIDATIVE)

+ -

Oxidase Oxidase

+ - + -

Vibrio spp. Aeromonas

Pleisiomonas

E.coli Klebsiella Enterobacter

Proteus

Citrobacter

Serratia

Salmonella Shigella

Proteus

Providentia

Edwardsiella

Yersinia

Pseudomonas aeruginosa

Burkholderia

Acinetobacter baumannii

Stenotrophomonas maltophilia

Neisseria

Morexella Acinetobacter spp. ie lwoffi

Other useful tests

• 1. oxidase test

• 2. oxidative fermentation (OF test)

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Glucose fermentation

+ -

Oxidase

+ -

Lactose ferment

+ -

OF glucose (OXIDATIVE)

+ -

Oxidase Oxidase

+ - + -

Vibrio spp. Aeromonas

Pleisiomonas

E.coli Klebsiella Enterobacter

Proteus

Citrobacter(with

H2S)

Serratia

Salmonella Shigella

Proteus (with H2S)

Providentia

Edwardsiella

Yersinia

Pseudomonas aeruginosa (motile)

Burkholderia mallei(non-motile)

Burkholderia pseudomallei(motile,

oxidize lactose)

Chromobacterium bividum (motile;

yellow pigment)

Flavobacterium (non-motile; yellow

pigment)

Acinetobacter baumannii-calcoaceticus (non-motile)

Stenotrophomonas maltophilia

Neisseria

Morexella (non-motile)

Alcaligenes (motile)

Acinetobacter spp. ie lwoffi

MacConkey: No Growth Gram Negative Bacilli

• Chocolate Agar Growth

– Neisseria

– Pleomorphic; require X+V factors or V factor

• Haemophilus

– X facter: hemin, V facter: NAD (staph)

• Chocolate Agar No Growth

– Need special environments or media

• Micro-aerophilic: Campylobacter, H. pylori, Arcobacter

• Rickettsia, Legionella, Brucella, Bartonella, etc

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Further Identification

A/A A/A H2S Alk/A

Alk/A H2S

Alk/Alk A/AG

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IMViC

Tryptophan, for example, is hydrolyzed to indole, pyruvic acid and ammonia by tryptophanase. Indole can be detected by reaction with Kovac's reagent (para-dimethylaminobenzaldehyde in alcohol) to produce a red color.

Methyl Red (MR) and Voges-Proskauer (VP) broth basis of acid or acetylmethyl carbinol production/Embden-Meyerhof pathway ?

Indole Test Citrate test

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IMViC

• ++-- E.coli

• --++ Klebsiella, Enterobacter

Motility test

Atrichous : Klebsiella Brownian Movement • A-Monotrichous; B-Lophotrichous; • C-Amphitrichous; D-Peritrichous.

Vibrio

E.coli

Campylobacter

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Urease test

Important features glucose non-fermenters

• Pseudomonas aeruginosa – Green pigment of pyocyanin, grape-like or tortilla-

like odor, metallic sheen

• Burkholderia pseudomallei – wrinkled colonies – Earthy odor – Oxidation of lactosePink colonies on McConkey

agar – Ashdown’s medium

• Acinetobacter baumanii – Growth at 42 c (ddx from calcoaceticus)

– non-motile, positive urease

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Lactose fermenter

• Proteus – Heavy H2S, swarming colonies

– IMViC +/–(negative in P.mirabilis)+,+/-,+

– Urease test: positive

• Escherichia coli : IMViC ++--, motile

• Serratia marcesens – Red pigment

• Klebsiella pneumoniae: IMViC --++, non-motile, mucoid colonies

• Shigella: Alk/A

• Salmonella: heavy H2S with gas – Exception: S.cholerae suis, S. paratyphi A: no H2S

:S. typhi: small H2S, no gas

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Vibrio

• V. cholerae: Beta-hemolysis, ferment sucrose

– Biotype: classic/eltor (mild, more carrier, pandemic)

– Serogroup: O1, O139

– Serotype: inaba, hikojima, ogawa

Haemophilus Organism

Infection Site

X factor

V factor

H. influenzae

Respiratory tract, meninges, blood, and other areas

+

+

H. aegyptius

Conjunctiva

+

+

H. haemolyticus

Respiratory tract (not pathogenic)

+

+

H. parainfluenzae

Respiratory tract (rarely pathogenic)

-

+

H. parahaemolyticus

Respiratory tract (not pathogenic)

-

-

H. ducreyi

Genital region

+

-

H. aphrophilus

Respiratory tract, blood, brain, others

+/-

-

Then, nitrocefin test to detect beta-lactamase

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Gram-Positive Rods

• Large regular rod, spore forming – Bacillus, Clostridium

• Small regular rod – Catalase positive: Listeria (ferment), Kurthia (oxidase)

– Catalase negative: Erysipelothrix (H2S), Lactobacillus (no H2S)

• Small irregular rod – Catalase positive: Corynebacterium,

Propionibacterium, Dermabacter, Rothia, etc – Catalase negative: Arcanobacterium, Leuconostoc

(vancomycin resistant; also Pediococcus, Lactobaphilus, Erysipelothrix),

Bifidobacterium, Gardnerella

Clostridium perfringens Bacillus cereus

Listeria monocytogenes : positive catalase : tumbling motility, beta-hemolysis( look alike GBS) : Umbrella-shaped motility : CAMP positive

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Other medias

• Mycobacteria

• Fungi

• Susceptibility