07/10/54

Bacterial Identification The Essentials for Plate Round

Rongpong Plongla, MD. MSc. Oct 07, 2011

Diagnosis of Bacterial Infection

microscopy unstained or stained with e.g.

Stain Decolorise Counterstain

culture identification by biochemical or serological tests on pure growth from single colony

on plates or in broth

sensitivities by disc diffusion methods, breakpoints or MICs Serodiagnosis DNA technologies

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Identification of

• Morphology • Growth requirements • Biochemistry • Enzymes • Antigens

Always Starting with Gram 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 – 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, aeruginosa, Serratia marcescens, Aspergillus, Nocardia, Burkhodelria cepacea

Staphylococcus

• 1. looking at 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 Susceptibility – S. saprophyticus: resistant to novobiocin – S. epidermidis: sensitive to novobiocin

S. saprophyticus caused UTI

Streptococci

• 1. looking at 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 Group B streptococci showing the – GBS classical “arrow-shaped hemolysis near the staphylococcus streak – Listeria ivanovii – Rhodococcus equi

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

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

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.

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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 enterococciusing 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 – 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 + Catalase -

Staphylococcus Streptococci

Hemolysis on sheep blood

Coagulase β α γ Bacitracin - (A-disc) + PYR* Optochin (P-disc) S R S S. aureus Staphylococcus R Coagulase-negative Strep gr.A S.pneumoniae Bile esculin Hippurate + -

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

+ -

6.5% NaCl, PYR Strep not gr. D (γ) + - Viridan Strep (α) Enterococci S. bovis

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Gram-Negative Organisms

• 1. Guessing from Gram Stain – Large gram-negative bacilli • , 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 – 10% of lactose – 10% 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 in the slant. • However, if either sucrose or lactose are fermented, sufficient products will be formed to turn both the butt and the slant yellow.

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• เมื่อ Inoculate เชื้อลงไปเชื้อที่ slant จะมีปริมาณมากกว่าบริเวณอื่น bacteria จะใช้น ้าตาล glucose หมดเร็วกว่า ถ้าเชื้อไม่สามารถใช้ lactose และ sucose ได้จะหันไปใช้ protein(polypeptone) ที่มีอยู่ใน media แทน ท าให้ slant มีภาวะ เป็นด่าง เปลี่ยน Phenal red เป็นสีแดง ส่วน bott นั้นมีแบคทีเรียที่เจริญอยู่น้อยกว่า slant ภายใน 24 ชั่วโมงจะ ยังใช้ glucose ไม่หมดจึงมีความเป็นกรดอยู่ (เปลี่ยนสี phenal red เป็นสีเหลือง) ปฏิกิริยาจึง เป็น K/A ( Slant/Butt) • ถ้าเชื้อสามารถใช้น ้าตาลได้ตั้งแต่ 2 ชนิดขึ้นไป จะท าให้อาหารทั้งหลอดเป็นสีเหลือง ปฏิกิริยาจึงเป็น A/A • ถ้าเชื้อไม่สามารถใช้น ้าตาลทั้ง 3 ชนิดได้เลย ปฏิกิริยาอาจเป็น N/N , K/N , A/N ( N คือไม่มี การเปลี่ยนแปลง ) • ถ้าเชื้อใช้น ้าตาลแล้วให้ gas แล้ว gas จะดันให้ media ยกตัวขึ้น หรือมีรอยแยกของ media • ถ้าเชื้อสร้าง H2S จะท าปฏิกิริยากับ Ferrous ion ที่มีอยู่ใน mediaได้ ferous sulfide ท า ให้เกิดเป็นตะกอนสีด าขึ้น

• When you see TSI you should mention – 1. Butt – 2. Slant – 3. Gas – 4. H2S Yellow=acid Pink=alkali ก้นเหลือง=ferment glucose เหลืองทั้งหลอด=ferment ทั้ง glucose+lactose+/-sucrose ไม่เหลืองเลย=glucose non-fermentors

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Glucose fermentation

+ -

Oxidase OF glucose (OXIDATIVE) + - + - Oxidase Oxidase Vibrio spp. Aeromonas Lactose ferment Pleisiomonas + - + -

+ - Pseudomonas aeruginosa Neisseria Acinetobacter Burkholderia Morexella spp. ie lwoffi

E.coli Salmonella Acinetobacter baumannii Klebsiella Shigella Stenotrophomonas maltophilia Enterobacter Proteus Proteus Providentia Citrobacter Edwardsiella Serratia Yersinia

Other useful tests

• 1. • 2. oxidative fermentation (OF test)

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Glucose fermentation

+ -

Oxidase OF glucose (OXIDATIVE) + - + - Oxidase Oxidase Vibrio spp. Aeromonas Lactose ferment Pleisiomonas + - + -

+ - Pseudomonas aeruginosa (motile) Neisseria Burkholderia (non-motile) Acinetobacter Morexella Chromobacterium bividum spp. ie lwoffi (motile; yellow pigment) (non-motile) Alcaligenes E.coli Salmonella Flavobacterium (non-motile; Klebsiella yellow pigment) (motile) Enterobacter Shigella Proteus Proteus (with H2S) Citrobacter(with Providentia Acinetobacter baumannii-calcoaceticus (non-motile) H2S) Edwardsiella Stenotrophomonas maltophilia Serratia Yersinia

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/AG A/A Alk/ H2S Alk/A A Alk/Alk H2S

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IMViC

Indole Test Methyl Red (MR) and Tryptophan, for example, is Voges-Proskauer (VP) hydrolyzed to indole, and ammonia by tryptophanase. Indole can be detected by reaction broth with Kovac's reagent (para- basis of acid or acetylmethyl carbinol production/Embden- dimethylaminobenzaldehyde in Meyerhof pathway ? alcohol) to produce a red color.

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IMViC

• ++-- E.coli • --++ Klebsiella, Enterobacter

Motility test

Vibrio

Campylobacter

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

E.coli

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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 lactosePink colonies on McConkey agar – Ashdown’s medium • Acinetobacter baumanii – Growth at 42 c (ddx from calcoaceticus) – non-motile, positive

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Lactose fermenter

• Proteus – Heavy H2S, swarming colonies – IMViC +/–(negative in P.mirabilis)+,+/-,+ – Urease test: positive • : 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 + + Then, nitrocefin test to detect beta-lactamase H. aegyptius Conjunctiva + +

Respiratory tract (not pathogenic) + + H. haemolyticus

H. parainfluenzae Respiratory tract (rarely pathogenic) - +

H. parahaemolyticus Respiratory tract (not pathogenic) - -

H. ducreyi Genital region + -

H. aphrophilus Respiratory tract, blood, brain, others +/- -

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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 Large gram+ bacilli

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

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