Question

Culture site: stitch abscess white, medium sized colony beta hemolysis catalase – positive slide coagulase – positive tube coagulase- positive A disk – negative CAMP test – negative Bile esculin – positive NaCl – positive Optochin disk – negative Bile solubility – negative PYR – negative id the organism

Answer 1

Pus, also known as purulent drainage, is a general term for a collection of thick, often white fluid that accumulates around the source of an infection. This can occur around many types of infections, but can be particularly alarming after surgery.

After a surgical procedure where an incision is made is is possible to have pus coming from the incision if an infection is present. While not all drainage is pus, drainage is often alarming because it can signal the presence of an infection, and can look very strange coming from a surgical incision.  

Blood agar is a rich, non-selective medium that supports the growth of most bacteria. However, it is differential based on the ability of the organism to produce enzymes called hemolysins, which lyse red blood cells (RBC). Three patterns of hemolysis can be observed on a blood agar plate.

1. Alpha ( a ) -hemolysis: incomplete lysis of RBC - Greenish, cloudy zone around the colony.

2. Beta ( b)-hemolysis: complete lysis of RBC - A clear zone with a clean edge around the colony.

3. Gamma ( g)-hemolysis: no lysis of RBC - No change in the blood agar around the colony.

MSA contains a high concentration of salt (NaCl), and therefore, selects for the growth of microbes that can tolerate high salt concentrations. Staphylococcus species are halotolerant, whereas Streptococcus species and many other organisms are inhibited by high concentrations of NaCl. MSA also differentiates on the basis of mannitol fermentation. Microorganisms that can ferment mannitol create acidic byproducts, which decrease the pH of the medium and cause the pH indicator, phenol red, to turn yellow.

Expected results:

Staphylococcus aureus

• Growth on MSA, ferments mannitol causing a color change to yellow
• b-Hemolysis on BAP

Staphylococcus epidermidis

• Growth on MSA, does not ferment mannitol (medium remains red)
• g-Hemolysis on BAP

Streptococcus pneumoniae

• Limited or no growth on MSA
• a-Hemolysis on BAP

Streptococcus pyogenes

• Limited or no growth on MSA
• b-Hemolysis on BAP

• The Tube Coagulase Test

  Principle:

  This method helps to measure free coagulase. The free coagulase secreted by S.aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.

  Procedure:

• Three test tubes are taken and labeled “test”, “negative control” and “positive control”.
• Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma.
• To the tube labeled test, 0.2 ml of overnight broth culture of test
• bacteria is added.
• To the tube labeled positive control, 0.2 ml of overnight broth culture of known S.aureus is added
• To the tube labeled negative control, 0.2ml of sterile broth is added.
• All the tubes are incubated at 37oC and observe the suspensions at half hourly intervals for a period of four hours.
• Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube.
• If the test remains negative until four hours at 37oC, the tube is kept at room temperature for overnight incubation.

• Applications:

• The coagulase test is used to distinguish between pathogenic and nonpathogenic members of the genus Staphylococcus. All pathogenic strains of S. aureusare coagulase positive whereas the nonpathogenic species(S. epidermidis) are coagulase negative.
• While slide coagulase test is useful in screening, tube coagulase test is useful in confirmation of coagulase test.
• Not all S.aureus strains produce coagulase; such rare strains are identified by thermonuclease test.

• Limitations:

• The slide test should be read very quickly, as false positives can occur.
• Auto agglutination may occur.
• Use water instead of saline for mixing.
• The slide test should not be performed with organisms taken from high-salt media such as Mannitol Salt Agar, as the salt content can create false positives.
• Over mixing may cause the clot to break down.
• The tube test is more reliable than the slide test.
• We generally don’t use the coagulase test when identifying unknowns.
• Samples must be observed for clotting within 24 hours. This is because some strains that produce coagulase also produce an enzyme called fibrinolysin, which can dissolve the clot. Therefore, the absence of a clot after 24 hours is no guarantee that a clot never formed. The formation of a clot by 12 hours and the subsequent disappearance of the clot by 24 hours could produce a so-called false negative if the test were only observed at the 24-hour time.

• CAMP test was first identified by Christie, Atkins, and Munch-Peterson in 1944 and CAMP test is an acronym of three researchers.

  CAMP (Christie, Atkins, and Munch-Peterson) test is used for the presumptive identification of Group B Streptococci (Streptococcus agalactiae). It is the only beta-hemolytic Streptococcus which secrete a protein called CAMP factor or “protein B”. CAMP test rarely give false positive with other Streptococci

  Variety of methods are currently available to identify Group B Streptococci (GBS) isolated from clinical specimens. The standard CAMP test and the CAMP spot test (rapid test) are mostly used. Standard camp test are time consuming and/or expensive compared to the CAMP spot test.

  Principle

  CAMP test detects the production of diffusible, thermostable, extracellular protein known as CAMP factor, produced by Group B Streptococcus. The CAMP factor acts synergistically with the beta lysin produced by Staphylococcus aureus to produce a zone of enhanced lysis of sheep or bovine erythrocytes. The standard CAMP test depend on the elaboration of two toxins during growth to form a typical arrowhead or flame-shaped clearing at the junction of the two organisms when they are placed perpendicular to each other.

  The rapid test utilizes an extract of Staphylococcal beta-lysin that acts directly with the CAMP factor previously diffused in the medium around the S. agalactiae colony. A positive CAMP reaction is indicated by an enhanced hemolysis within 30 minutes to 1 hour of adding a drop of CAMP factor reagent

  Bile Esculin agar (BEA) is used for this test. BEA is a selective and differential medium which is presumptively used to identify Enterococci and group D Streptococci based on the ability of an organism to hydrolyze esculin. Bile esculin agar contains oxygall (bile salts, first selective ingredients) to inhibit the growth of other gram positive organisms other than Enterococci and group D Streptococci. It contains sodium azide (second selective ingredients) to inhibit the growth of gram negative organisms. It also contains nutrients esculin and ferric citrate. Esculin (differential ingredient) is a fluorescent compound and its hydrolysis can be observed by a loss of fluorescence.
  Bile esculin disk is used for the rapid detection of esculin hydrolysis in presence of bile for differentiating group D Streptococci from non-group D Streptococci.

  Principle

  Bile esculin test is based on the hydrolysis of esculin into glucose and esculetin (6, 7-dihydroxy-coumarin) by a micro-organism that produce an enzyme esculinase. Esculetin reacts with an iron salt (ferric citrate) in the medium to form a phenolic iron complex which produces a dark brown or black color.

  Esculin + Acid        → β-D-glucose + Esculetin

  Esculetin + Fe3+ → Dark Brown Color

  Procedure

  A. Tube Test

• Using sterile loop, pick one or two colonies from an 18-24 hours culture.
• Inoculate onto the surface of slant of bile esculin medium with an S-shaped motion.

• B. Disk Test

• Moisten disk with a single drop of distilled water.
• Using a sterile loop, pick two or three well-isolated colonies from an overnight culture.
• Inoculate on the disk.
• Allow to react at room temperature for 10min.
• Observe the disk for the development of a dark brown or black color.
• Incubate the inoculated tube at 35-37°C for 24 hours.
• Observe the result.

  Result-

  Positive: Blackening of more than half of the agar slant, if the medium containing ferric ammonium citrate     is used.
  NOTE: In case of esculin broth without iron citrate, positive test means blackening of the broth after addition of the ferric reagent or by the loss of fluorescence of the medium.

  Negative: No blackening of medium.

                      OR If the agar slant is less than half darkened or medium will fluoresces under UV light.

  Positive Disk Test: Development of a dark brown or black color.

  Negative Disk Test: Remains colorless

• Principle of Bile Solubility Test

  Bile or a solution of a bile salt (e.g., sodium desoxycholate) rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme, amidase. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process.

  Procedure of Bile Solubility Test

• After 12 to 24 hours of incubation on 5% sheep blood agar, place 1 to 2 drops of 10% sodium desoxycholate on a wellisolated colony. Note: A tube test is performed with 2% sodium desoxycholate.
• Gently wash liquid over the colony without dislodging the colony from the agar.
• Incubate the plate at 35°-37°C in ambient air for 30 minutes.
• Examine for lysis of colony.

• Limitations of Bile Solubility Test

  Enzyme activity may be reduced in old cultures. Therefore, negative results with colonies resembling S. pneumoniae should be further tested for identification with alternate methods.

  Result Interpretation of Bile Solubility Test

  Positive: Colony disintegrates; an imprint of the lysed colony may remain in the zone.
  Negative: Intact colonies.

• PYR (Pyrrolidonyl Aminopeptidase) Test is used for the detection of pyrolidonyl arylamidase (also called pyrrolidonyl aminopeptidase) activity in Streptococcus pyogenes (group A strep), Enterococcus spp., some coagulase-negative staphylococci, and some Enterobacteriaceae. It is also known as PYR (L-pyrrolidonyl-β-naphthylamide) which serve as a substrate for the detection of pyrrolidonyl peptidase.

  Facklam, Thacker, Fox and Eriquez reported that 98% of group A streptococci and 96% of group D enterococci hydrolyze PYR. Although Aerococcus species are rarely isolated in the clinical laboratory, these organisms are also expected to hydrolyze PYR.

  Facklam et al. further reported that 98% of group B streptococci, 100% of non-group A, B and D streptococci, 100% of group D non-enterococci and 82% of viridans streptococci yield negative PYR test results.

  Principle of PYR Test

  PYR is a rapid method for presumptive identification of bacteria based on the pyrrolidonyl arylamidase enzyme. The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl- β-naphthylamide substrate to produce a β-naphthylamine. The β-naphthylamine can be detected in the presence of N,N-methylaminocinnamaldehyde reagent by the production of a bright red precipitate.

  Following hydrolysis of the substrate by the peptidase, the resulting b-naphthylamide produces a red color upon the addition of 0.01% cinnamaldehyde reagent. When a visible inoculum of microorganism is rubbed onto a small area of a disk impregnated with the substrate, the hydrolysis occurs within 2 min, at which time the cinnamaldehyde reagent is added to detect the reaction by a color change to purple.

  Uses of PYR Test

• It is used for the presumptive identification of group A streptococci (Streptococcus pyogenes).
• It is used for the rapid differentiation of enterococci from group D ß-hemolytic streptococci.
• It also differentiate some Staphylococci (positive haemolyticusfrom negative S. auricularis).
• It is used in the identification of E. coli, separating it from other indole positive, lactose positive, gram-negative rods.
• Gram-negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain protocol. Gram-negative bacteria will thus appear red or pink following a Gram stain procedure due to the effects of the counterstain (for example safranin).

  The Gram Stain

  In microbiology, the visualization of bacteria at the microscopic level is facilitated by the use of stains, which react with components present in some cells but not others. This technique is used to classify bacteria as either Gram-positive or Gram-negative depending on their colour following a specific staining procedure originally developed by Hans Christian Gram. Gram-positive bacteria appear dark blue or violet due to the crystal violet stain following the Gram stain procedure; Gram-negative bacteria, which cannot retain the crystal violet stain, appear red or pink due to the counterstain (usually safranin).

  The reason bacteria are either Gram-positive or Gram-negative is due to the structure of their cell envelope. (The cell envelope is defined as the cell membrane and cell wall plus an outer membrane, if one is present.) Gram-positive bacteria, for example, retain the crystal violet due to the amount of peptidoglycan in the cell wall. It can be said therefore that the Gram-stain procedure separates bacteria into two broad categories based on structural differences in the cell envelope.

  Characteristics of Gram-negative Bacteria

  Gram-negative bacteria have a characteristic cell envelope structure very different from Gram-positive bacteria. Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the loose network of peptidoglycan chains referred to as the peptidoglycan layer.