VPM 201: Veterinary Bacteriology & Mycology, 2012

Laboratory 3a & b. Gram-positive cocci: Staphylococci, Streptococci & Enterococci

A. Detailed flowcharts summarizing major Genera & species.

1. Catalase-positive cocci.

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2. Catalase-negative cocci.

G-positivecocci

CatalaseTest

Enterococci

Negative

LancefieldGrouping

Streptococci

16s rDNA

Group C

beta hemolysis= alpha hemolysis (zone-of-greening)nonhemolytic

People StrepsS. pyogenes Group A

*S. pneumoniae

"-" or "+" indicate ability to fermentTrehalose/Sorbitol respectively

Esc = Positive Esculin hydrolysis

Group D

Canine Strep(s)S. canis

Positive

Staphylococci&

Micrococci

Group G

Bovine StrepsS. agalactiae CAMP+ve

S. dysgalactiae *S. uberis Esc

Group B & C

Equine Streps - S. equi (- / -)

S. zooepidemicus (- / +)*S. equisimilis (+ / -)

Porcine Streps*S. suis

*S. porcinus

* =Variable or no Lancefield Group

E. faecalis EscE. faecium Esc

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B. Outline of primary culture (colony) assessment steps. [modified from L.S. Garcia (editor-in-

chief), Clinical Microbiology Procedures Handbook, 3rd edition. ASM press, 2007, pp. 3.3.2.1-3.3.2.5.]

1. Examine plates closely for growth and distinct colony types. A magnifying lens may be

required to see very small colonies.

2. Record information on a “Diagnostic Worksheet” and use simple “tracking” terminology such

as Col # 1, Col # 2 for distinct colony types. It is not unusual for a single Streptococcal isolate to

display two distinct colony types on the same plate.

3. Indicate relative colony numbers (growth).

No growth (NG):

Scant growth (SG): 1 – 5 colonies (are these colonies along streak lines or contaminants?)

Few: Growth primarily in first quadrant.

Moderate growth (MG): First & second quadrants.

Heavy growth (HG): Third or fourth quadrant growth.

4. Use gross colonial morphology terminology introduced in Lab 2.

5. Assess colonies for evidence of hemolysis.

- α (alpha)-hemolysis – “zone of greening” resulting from conversion of hemoglobin to

methemoglobin adjacent to, or beneath, bacterial colonies. Good examples include Strep.

pneumoniae & Strep. dysgalactiae.

- β (beta)-hemolysis – generally a wide, clear, single-zone of hemolysis around colonies.

Good examples include the “equine” streps: Strep. equi subsp. equi and zooepidemicus.

Note: There are β-type “variants” that are typical for a given species. A good example is

Streptococcus agalactiae.

- Double-zone (or “target-hemolysis”) is often seen with Staph. aureus & Staph.

intermedius Group colonies.

C. Things in your Laboratory Handbook that you may find helpful include:

1. Gram-positive flowchart (cocci section) page 5.

2. CAMP, Catalase, Coagulase and Carbohydrate fermentation test descriptions in

Appendix C.

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General Worksheet (modified from AVC Diagnostic Bacteriology 27-8-12)

Exercise 1: Gram-stain (Direct Smear) : _not available _______________________________________

1Media 2Colony Characteristics Gram-stain 3Additional Tests Tentative i.d.

BA

Moderate size (1.0 mm), circular, convex, off-white, opaqueDouble-zone (target) hemolysis

Gram-positive cocciClumps, pairs, occasional short (3-4) chains

Catalase (CAT) +ve

Coagulase +ve

Staph. intermedius GroupOr

Staph. pseudintermedius Complex

OrStaph. pseudintermedius

MACNG Generally not done

from MAC colonies.

1. see Laboratory Handout manual for media descriptions. 2. Relative amount of colony type(s): Hg/Mg/Sg/Ng – Heavy, Moderate, Few/Scant and No growth, respectively. 3. Examples include; TSI, SIM, Urea, Citrate, Indole, CO, CAT, Coagulase (see Lab Handout for test descriptions).

Exercise 2: Gram-stain (Direct Smear): _not available _______________________________________

1Media 2Colony Characteristics Gram-stain 3Additional Tests Tentative i.d.

BA

Pinpoint to small (<0.5 mm), circular, translucent“subtle” β-type hemolysis

Gram-positive cocci, smaller than Exercise 1Clumps, short chains

CAT –ve

CAMP test set upMastitis Strep. spp

Small (<0.5 mm), circular, translucentα-type hemolysis

Gram-positive cocci, smaller than Exercise 1Clumps, short chains

CAT –ve

CAMP test set upMastitis Strep. spp

MACNG Generally not done from

MAC colonies.

Exercise 3: Gram-stain (Direct Smear): _not available _______________________________________

1Media 2Colony Characteristics Gram-stain 3Additional Tests Tentative i.d.

BA

Moderate size (1.0 mm), circular, convex, off-white, opaqueNo hemolysis

Gram-positive cocciClumps, pairs, occasional short (3-4) chains

CAT +ve

Coagulase +veStaph. hyicus

MACNG Generally not done from

MAC colonies.

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D. Lab 3a: Student Exercises

For each of your exercises use culture, Gram-stain and test results, as well as colony and microscopic observations, in the context of the animal(s) involved and clinical presentation, to tentatively identify pathogens. The Gram-positive cocci flow charts in Part A of this lab can help.

Exercise 1. A dog presents with pyoderma of the face and limbs. Material from pustules was plated on Blood (BA) and MacConkey (MAC) agars and incubated overnight at 37 ºC. BA plates are provided - there was no growth on MAC.

a. Use the guidelines in Part B to assess any colonies that might be present on your plates. Use the worksheet on the opposite page to record information.

b. Is there hemolysis? Is there an obvious type? Recall that “double-zone” hemolysis (target-hemolysis) is generally limited to several coagulase-positive Staphylococci (ex. S. aureus, S. intermedius Group) and Clostridium perfringens (gram-positive bacilli, spore-forming, obligate anaerobe).

c. Do a Gram-stain on distinct colony types that might be present. Examine and record your results on the worksheet.

d. Perform a catalase test. Is it positive or negative? Positive

e. Is your colony coagulase positive or negative? (see Demo 2 – Exercise 1). Positive

Exercise 2. The colonies on BA are from a Holstein with acute mastitis. The organism did not grow on MAC. Please perform the following tests.

a. Use the same process from Exercise 1 to assess your plate.

b. Set up a CAMP reaction on a BA plate provided – an instructor will provide you with a procedure. Each student should make an attempt.

Exercise 3. Colonies growing on BA are from an acute exudative dermatitis in a recently weaned pig. The plates were incubated at 37 ºC for 24 hrs. There was no growth on MAC.

a. Use the same process from Exercise 1 to assess your plate. .

b. Is this pathogen coagulase positive? (see Demo 2- Exercise 3). This strain is positive

E. Demonstration Materials

1. Bacterial cultures on BA plates: S. aureus, S. intermedius Group & S. hyicus

2. Coagulase tests

a. Pathogens from Exercise 1 & 3.

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Exercise 1: Gram-stain (Direct Smear) : _not available _______________________________________

1Media 2Colony Characteristics Gram-stain 3Additional Tests Tentative i.d.

BA

Small (<0.5 mm), circular, translucent, shiny/mucoid “dew-drop”β-type hemolysis

Gram-positive cocci, smaller than Exercise 1

Clumps, short chains

CAT –ve

Note: Sugar fermentation will distinguish – trehalosi, sorbitol

“Equine Strep”- S. equi subsp. equi- S. equi subsp. zooepidemicus- S. dysgalactiae subsp. equisimilis

MACNG Generally not done from

MAC colonies.

1. see Laboratory Handout manual for media descriptions. 2. Relative amount of colony type(s): Hg/Mg/Lg/Ng – Heavy, Moderate, Low and No growth, respectively. 3. Examples include; TSI, SIM, Urea, Citrate, Indole, CO, CAT, Coagulase (see Core lab manual for test descriptions).

Exercise 2: Gram-stain (Direct Smear): _not available _______________________________________

1Media 2Colony Characteristics Gram-stain 3Additional Tests Tentative i.d.

BA

see Exercise 2, Lab 3A(subtle – β-type hemolysis)

see Exercise 2, Lab 3A CAMP +ve“arrowhead zone”

Strep. agalactiae

see Exercise 2, Lab 3A(α-type hemolysis)

see Exercise 2, Lab 3A CAMP –ve Strep. dysgalactiae

MACNG Generally not done from

MAC colonies.

Exercise 3: Gram-stain (Direct Smear): _not available _______________________________________

1Media 2Colony Characteristics Gram-stain 3Additional Tests Tentative i.d.

BA

Pinpoint, circular, shiny,Faint α-type hemolysis developing.

Note: Colony variation is a feature of a number of Strep. spp.

Gram-positive cocci,Pairs with an elongated

(lancet) morphology CAT –ve Strep. suis

MACNG Generally not done from

MAC colonies.

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Lab 3b: Gram-positive cocci

A. Student Exercises.

Use the same guidelines and support information utilized for exercises yesterday to work though these cases and tentatively identify the most likely pathogen.

Exercise 1: This organism was isolated from a 2-year old horse with a nasal discharge and fever.

The horse had difficulty breathing and swallowing. The organism did not grow on MAC.

a. Use the worksheets on the opposite page to record your results.

b. What is your tentative identification and why? animal, clinical problem, culture, gram-stain & tests consistent with i.d. (see sheet)

Exercise 2: Examine your CAMP test results that you set up from Exercise 2 yesterday.

a. Is your organism CAMP-positive, Camp-negative or CAMP-inhibitory? see worksheetb. Compare your results to an adjacent group. c. What is happening when an organism is CAMP positive? Synergistic hemolysis between test-bug & S. aureus (in this case) outer-zone hemolysin.d. See the CAMP tests (Demo 2). e. Using your CAMP results and information gathered from your primary plate yesterday you should be able to identify this pathogen to the species level. What is it and why? animal, clinical problem, culture, gram-stain & tests consistent with i.d. (see sheet)

Exercise 3. Joint fluids from a few slaughter hogs showing signs of arthritis and/or valvular

endocarditis were collected using sterile swabs and submitted for standard aerobic culture.

BA and MAC plates were inoculated and gown overnight at 37 oC. There was no growth on

MacConkey’s.

a. Use the worksheets on the opposite page to record your results.

b. What is your tentative identification and why? animal, clinical problem, culture, gram-stain & tests consistent with i.d. (see sheet)

B. Demonstration materials

1. Cultures: S. agalactiae, S. dysgalactiae, S. equi, S. suis

2. CAMP test reactions: S. agalactiae, S. dysgalactiae and S. uberis

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C. Questions

1. What simple test will distinguish Staphylococci & Streptococci taken from pure colonies?

Answer - Catalase test

2. Is Gram-staining and microscopic examination a reliable technique for distinguishing

Staphylococci & Streptococci?

From colony material? (Y/N) – generally no

From clinical samples? (Y/N) – liquid samples ( exudate, pus, milk) are more reliable

3. Historically the “coagulase test” involved a “rapid” slide test and a “slower” tube test when

assessing Staphylococci? We now know that one test measures a “true” secreted-coagulase while

the other measures a cell-associated Clumping Factor (ClfA). Indicate which does what below.

Rapid Slide test: ClfA – agglutination occurs by binding fibrinogen

Tube Test: Coagulase – a clot forms by polymerization of fibrinogen to fibrin

4. In what disease conditions are staphylococci commonly associated with in:

Dogs: SSTI’s (Skin + Soft Tissue Infections), otitis externa, UTI, reproductive etc..

Currently, S. pseudintermedius (of the S. intermedius group) and to a lesser extent S.

schleiferi subsp..

Cows: S. aureus - mastitis, pustular dermatitis on udder (impetigo), CoNS – mastitis.

Pigs: S. hyicus - young pigs (dermatitis, septicemia, arthritis); S. aureus – mastitis,

udder impetigo, endometritis in sows.

Poultry: S. aureus – Bumblefoot, septicemia & arthritis in turkeys

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5. Name the major disease conditions caused by the species of Streptococci dealt with today?

Streptococcus agalactiae – mastitis in ruminants

Streptococcus dysgalactiae subsp. dysgalactiae (Strep. dysgalactiae) – mastitis in ruminants

Streptococcus equi subsp. equi – Strangles (submandibular, retropharyngeal lymphadenitis

with abscessation) and/or Bastard Strangles (metastatic abscessation to: mediastinal lymph

nodes, lungs, liver, spleen, brain, guttural pouch – from retropharyngeal L.N.).

Streptococcus suis – septicemia, bronchopneumonia, endocarditis, arthritis, meningitis in

pigs.

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