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Group B Strep Prevention (GBS, baby strep, Group B streptococcal bacteria)

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Close up picture of GBS Colony

This is a close-up picture of a group B Streptococcus (GBS) growing on a 5% sheep blood agar plate. The red background is the blood agar plate and the gray circular dots are colonies of GBS growing on the agar. The colonies of GBS are gray to whitish-gray surrounded by a weak zone of hemolysis of the red blood cells in the culture medium. Hemolysis is one of the traits that is used to help identify the bacteria. Hemolysis is the destruction of the red blood cells suspended in the agar medium. Before performing the tests in the following photos, a gram strain and catalase test should be performed. GBS are gram-positive cocci in chains and catalase negative.


Comparison of GBS & GAS Hemolysis This is a picture of an agar plate with growth and hemolytic activity of group A streptococci (on the left) and group B streptococci (on the right). Note that the colonies of both bacteria appear about the same color and size. The difference between these two bacteria is in the degree of hemolytic activity. The group A Streptococcus has more hemolytic activity than the group B Streptococcus. Experienced microbiologist use these traits to identify the two types of bacteria.

Comparison of Hemolysis Group A,B,G, F Strep This is a picture of another blood agar plate. In this picture, the group A Streptococcus is in the quadrant on the top portion of the agar plate; the group B Streptococcus is shown on the right quadrant; a group G Streptococcus is shown on the bottom quadrant; and a group F Streptococcus is shown on the left quadrant. Although the differences may not be apparent to the untrained eye, there are small differences in the size of the hemolytic activity of each of these different streptococci, especially in the stabbed portion of the agar plate as depicted by the arrows. The GBS is the bacteria that causes the least amount of hemolytic activity. And the group F Streptococcus has the smallest colony size.

Slides of  Agglutination

The most accurate way to identify a group B Streptococcus is to demonstrate that the bacteria in question has the Lancefield group B antigen on the surface of the bacteria. The Lancefield antigens are called group A, B, C, D, etc. Each of the antigens is associated with a certain kind of bacteria. In the case of GBS, the antigen we are looking for is called group B. This picture shows a slide agglutination test that is used to identify the group antigens of streptococci. There are two different commercial products shown in this picture, the top two circles are one test and the bottom two circles are another test. The general procedure, for this test, is to make a small suspension of bacteria from the blood agar plate with a bacteriologic loop of culture and place it on the test card shown above. The suspension is placed in both circles. Group specific antibodies from the commercial grouping kit are then placed on the suspension. Next, the suspension of bacteria is mixed with the group specific antibody and tilted back and forth for about one minute. In this case, we put group B antibodies in the circles on the left and group A antibodies in the circles on the right. A positive agglutination reactions are shown on the left (both commercial tests). Note the clumping of the suspension of bacteria. A negative reaction is shown on the right. The suspensions mixed with the group A reagent are smooth; no clumps are apparent. These results indicate we have identified a group B Streptococcus.


CAMP Test Comparison If the laboratory is not able to identify group B streptococci by the Lancefield grouping procedure, there are other microbiologic tests that can be used to identify GBS. This picture shows one of these tests. It is called the CAMP test. CAMP is an acronym for the authors of this test (Christie, Atkinson, Munch, Peterson). The CAMP test takes advantage of the capacity of GBS to produce this CAMP factor; most other hemolytic streptococci do not produce CAMP factor.
This picture shows the growth and CAMP test of a group B Streptococcus (on the left) and a group A Streptococcus (GAS) (on the right). On the top of the agar plate we have inoculated the plate with a Staphylococcus strain (horizontal streak). We then inoculated the GBS (on left) and GAS (on right) perpendicular to the Staphylococcus streak. We inoculated the agar plate so as not to touch the two different organisms (Staphylococcus and Streptococcus) but to come close to each other. The Staphylococcus is used because it produces a lysin that only partially lyses the red blood cells (called beta-lysin). The CAMP factor reacts with the partially lysed area of the blood agar plate to enhance the hemolytic activity. Note the arrowhead shape of the zone of enhanced hemolytic activity by the GBS near the Staphylococcus streak (on left) but not by the GAS (on right). This means that the bacterium on the left is GBS because it is producing a CAMP factor.
The test shown on the bottom of this picture with the two small disks is the bacitracin sensitivity test. A positive zone of inhibition around the disk and a negative CAMP test shown in the agar plate on the right is a presumptive identification of a group A streptococcus.

Commercial Betalysin CAMP Test This picture shows a variation of the CAMP test. We have placed a disk at the top of this plate instead of the Staphylococcus streak. The disk contains the beta-lysin that is normally produced by the Staphylococcus. Since the disk is round, the shape of the increased hemolysis caused by the CAMP factor is different. These disks are available from several sources. Similar to the previous agar plates these tests were set up by: inoculating the plates, placing the disks in place, and incubating the plates overnight in an ambient atmosphere at 35oC.

Hippurate Tests This test is an additional presumptive test used to aid in the identification of GBS. This test, the test for free benzene, is one variation of the hippurate hydrolysis test. Hippurate (hippuric acid) is a benzene (6 carbon) ring with the amino acid serine attached to one of the carbon molecules. Hippuricase, the enzyme, cleaves the benzene ring and the serine free from each other. The streptococcal cultures are inoculated into tubes containing hippuric acid and incubated overnight (the two larger tubes on the left). The next day the tube with growth is centifuged to sediment the bacteria. A small portion of the broth is pipetted into small tubes (two small tubes on the right). A reagent called ferric chloride is added to both tubes. If a strong precipitate forms and remains after 10 minutes, the test is positive for hippurate hemolysis. In the picture on this page, reading from left to right, the third tube (first small tube) is positive because it has remained cloudy. The forth tube (second small tube) is still clear, therefore it is negative.

Hippurate Tests This test is an additional presumptive test used to aid in the identification of GBS. It is a variation of the hippurate hydrolysis test. Hippurate (hippuric acid) is a benzene (6 carbon) ring with the amino acid serine attached to one of the carbon molecules. Hippuricase, the enzyme, cleaves the benzene ring and the serine free from each other. In this case, some of the growth on a blood agar plate is suspended in a solution of hippurate and incubated for 4 hours. Next, a reagent ninhydrin is added. If a purple color develops, in the tube, the test is positive. The test detects the amino acid serine that has been cleaved from the hippuric acid. The second tube is positive for hippurate hemolysis and presumptive identification of GBS.

Antimicrobial Susceptibility Test Depicted above are two methods for determining antimicrobial susceptibility of GBS. It is especially important that the microbiologist determine the susceptibility of GBS to erythromycin and clindamycin as these two drugs are used as substitutes if the patient cannot be treated with penicillin.
The long strip on the left is called the E-testTM. This pictures shows the drug azithromycin (this is shown as an example: we recommend using an erythromycin or clindamycin E-testTM). In this test, the strip contains a gradation of antibiotic with the strongest being at the top of the strip and the weakest at the bottom. The minimum inhibitory concentration of antibiotic that will inhibit the bacteria is determined by where the growth of the bacteria starts, or the area of the growth where the ellipse growth meets the strip.
The two disks on the right of the agar plate are erythromycin and tetracycline (shown here as an example: we recommend using clindamycin for GBS). There are criteria for the zones of inhibition of growth that determine whether or not the bacteria is susceptible or resistant. In the test shown above, the bacterium is susceptible to erythromycin (the zone is large >26mm) and resistant to tetracycline (zone is small <12mm). Since the disk test predictability is accurate and considerably less expensive than the e-test, in most situations, the disk test is perfectly satisfactory for determining the antimicrobial susceptibility of GBS. Users should consult the NCCLS document below for interpretation of MIC’s or zones for determining susceptible intermediate resistance or resistance.

NCCLS Performance Standards for Antimicrobial Susceptibility Testing; Twelfth Informational Supplement: M100-12 (2002) M2-A7 and M7-A5.

Antimicrobial Susceptibility Test This test used to test all the ABC isolates. This picture shows the most accurate method of determining the antimicrobial susceptibility of GBS. This test is called the broth dilution antimicrobial susceptibility test. It is standardized by the the National Committee on Clinical Laboratory Standards (NCCLS). The picture above shows a microtiter plate with 96 wells. There are 8 rows of wells, labeled A through G. Each row of wells contains one antibiotic. In each row there are 12 wells. Each well contains a different concentration of the specific antibiotic, usually doubling dilutions. In this case, row A contains penicillin starting at the right at 16 ?g/ml and reducing to 0.008 well 12. Row B contains ampicillin starting in well 1 with 16 ?g/ml and reducing to 0.008 ?g/ml to well 12 in the same row. The other rows contain different antibiotics. All 96 wells are inoculated at the same time and the panel is incubated overnight (20-24h). The next day the wells are examined for growth. If there is growth in the well, the antibiotic did not inhibit the bacteria. If you take a close look in row A you will see growth (a small white button at the bottom of the well) only in well 12. In row B you see growth in both well 11 and 12. This means the minimum inhibitory concentration (MIC) of penicillin is 0.016 ?g/ml and the MIC for ampicillin is 0.032 ?g/ml. The MIC’s of these antimicrobials are considered susceptible for,GBS. Erythromycin is shown is row F. The concentration of erythromycin starts at 32 ?g/ml in well 1 and goes to 0.016 ?g/ml in well 12. You can see that there is growth in wells 12 to 4, meaning the MIC of erythromycin to this strain is 8 ?g/ml. This MIC is considered resistant for GBS. Since this is a standardized test results can be compared world-wide if the standard procedures are used. Consult the NCCLS manual for reporting interpretive standards (sensitive, intermediate, and resistance).NCCLS Performance Standards for Antimicrobial Susceptibility Testing; Twelfth Informational Supplement: M100-12 (2002) M2-A7 and M7-A5.



Page Last Modified: April 20, 2008
Content Last Reviewed: April 20, 2008
Content Source: National Center for Immunization and Respiratory Diseases
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