# Presentation: 661903

## Perinatal Group B Streptococcal Disease: Procedures for Collection and Processing Clinical Samples and Susceptibility Testing An Overview of Revised CDC prevention guidelines (2002)

**National Center for Infectious Diseases**
**Division of Bacterial and Mycotic Diseases**

**Notes:**
Title :  Perinatal Group B Streptococcal Disease:  Background, Epidemiology, and Overview of Revised CDC prevention guidelines (2002), Procedures for Collection and Processing Clinical Samples and Susceptibility Testing
Presented by: Centers for Disease Control & Prevention, National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases

## GBS Maternal Colonization

**GBS Carriers**
**10% - 30% of women**
**higher in African Americans and nonsmokers**
**clinical signs not predictive**
**dynamic condition**
**When to culture?**
**culture at delivery -- too late**
**prenatal cultures predict delivery status**

**Notes:**
GBS MATERNAL COLINIZATION:
Studies show that between 10 and 30 percent of women are colonized with GBS.
Rates are higher among African Americans and among nonsmokers.
GBS colonization in the gastrointestinal and genital tracts of women is an asymptomatic condition that does not result in symptoms and is not harmful in nonpregnant women.
Cannot determine GBS colonization status by taking a history or doing a physical exam, have to culture the bacteria from a clinical sample.
GBS colonization is a dynamic condition; GBS colonization may come and go over months.
Cultures done within 5 weeks before delivery date are highly predictive of colonization status at the time of delivery.
Intervals greater than 5 weeks between culture date and delivery are less accurate at predicting the mother’s GBS status. Yancey et al., OB GYN 1996;88:811-5.

## Predictive Value of Antenatal Cultures by Interval to Delivery

**N=826; 26.5% GBS carriers**
**Yancey et al., OB GYN 1996;88:811-5.**

_**>**_

**Notes:**
PREDICTIVE VALUE OF ANTENATAL CULTURES BY INTERVAL TO DELIVERY
Slide summarizes information regarding antenatal culture results collected by Yancey and colleagues from over 800 women delivering at the Tripler Army Medical Center.
Depicts the ability of GBS cultures collected prenatally to predict the culture status of women at the time of delivery.
Evident that test performance is significantly better for cultures collected within 5 weeks of delivery compared with those collected at longer intervals before delivery.
CDC guidelines recommend collecting cultures at 35-37 weeks gestation, when the accuracy of the test should be good.  Since some preterm deliveries will occur before the results of the late gestation culture are available, an empiric approach is recommended for preterm deliveries without culture results.  The new guidelines suggest intrapartum antibiotics for preterm deliveries (unless a culture was already obtained and known to be negative for group B streptococcus).

## Sensitivity and Specificity of Late Antenatal Cultures

_**>**_

**N=826**
**Yancey et al., OB GYN 1996;88:811-5.**

**Notes:**
SENSITIVITY AND SPECIFICITY OF LATE ANTENATAL CULTURES
This slide presents other test performance parameters from the same study by Yancey.
The sensitivity of late antenatal cultures for identifying colonization status 6 or more weeks before delivery was only 43%, while the specificity was 85%.
Cultures done between 1 and 5 weeks before delivery had a sensitivity of 89% and a specificity of 97%.
Sensitivity & specificity were calculated using results at the time of labor as the gold standard.

## GBS Carriage by Culture Site

*** P <0.05 both vs. vaginal only**

**Notes:**
GBS CARRIAGE BY CULTURE SITE
The site of culture is very important to increase the yield.
This slide illustrates the difference in yield if only one site - either the vagina or rectum – is cultured compared with culturing both sites.  
A significantly higher proportion of women are found to be GBS carriers if both sites are cultured.
In all studies, women enrolled received both a vaginal and a rectal swab.  The first row in the table shows results of a study of 789 women :
When vaginal swabs alone were analyzed, 10% of women tested positive for GBS carriage
When rectal swabs alone were analyzed, 18% of women tested positive for GBS carriage
When both rectal and vaginal were analyzed, 21% of women tested positive (significantly higher than either vaginal or rectal alone)
The second row of the table shows results of a study of 94 women:
When vaginal swabs alone were analyzed, 18% of women tested positive
When rectal swabs alone were analyzed, 28% of women tested positive
When both vaginal and rectal swabs were analyzed, 31% of women tested positive (significantly higher than either vaginal or rectal alone).
The last study in the last row of the table consisted of 301 women
11% of vaginal swabs tested positive
14% of rectal swabs tested positive
When both vaginal and rectal swabs were analyzed, 18% of women tested positive (significantly higher than either rectal or vaginal alone)  

## GBS Carriage by Culture Method

*** P <0.05 selective broth vs. blood agar**

**Notes:**
GBS CARRIAGE BY CULTURE METHOD
The method of culture is also very important.
Some labs simply plate swab on blood agar plate, incubate for 24 hours, and discard if no growth of GBS is identified.
This slide shows that if swabs are placed in selective broth medium first and incubated in medium for 18-24 hours before plating to blood agar, there is much higher yield of GBS

## The 2002 CDC Group B Strep Prevention Guidelines for Culture Collection & Processing. . .

**Notes:**
The 2002 CDC Group B Strep Prevention Guidelines for Culture Collection & Processing. . .
The upcoming slides describe CDC’s recommendations for culture collection and processing.

## Correct Methods for Specimen Collection & Handling are Important . . .

**Correct laboratory processing of culture specimens critical in screening strategy**
**Detailed and updated instructions on specimen collection, culture processing, and susceptibility testing provided in the 2002 revised guidelines**
**Universal group B strep recommendation – labs play increased role in prevention**

**Notes:**
SPECIMEN COLLECTION & HANDLING
Correct laboratory processing of culture specimens critical in screening strategy
Detailed and updated instructions on specimen collection, culture processing, and susceptibility testing provided in the 2002 revised guidelines
Labs will play a vital role in implementing the revised guidelines because they will see increased activity due to a recommendation for GBS universal screening. Therefore, it is important for labs to thoroughly understand their role in this process.

## CDC’s Recommendations for Prenatal Group B Strep Cultures

**Optimize cultures:**
**Site: vagina and rectum**
**single swab or two swabs**
**through anal sphincter**
**Timing: 35 to 37 weeks**
**Collection: NOT by speculum**
**self collection an option**

**Notes:**
CDC’S RECOMMENDATIONS FOR PRENATAL GBS CULTURES
During prenatal group B strep screenings, every effort should be made to optimize the yield from the cultures.
Includes culturing both vagina and rectum at the appropriate time
Either a single swab or with two swabs; if two are used, both can go into single selective broth culture.
Specimens should come from lower third of the vagina, and rectal swab should pass through the anal sphincter.
These cultures should be done at 35 to 37 weeks.
In the outpatient setting, self-collected cultures from both vaginal and rectal sites can be alternative to sample collected by clinicians, and patients may prefer to be offered this option.

## Inoculate swabs into nonnutritive transport medium
Appropriate transport systems are commercially available (e.g., Amies’ or Stuarts’)
Specimens in transport media will remain viable for up to 4 days
Specimens should be labeled clearly (i.e. GBS or pen allergy)

**Appropriate transport systems are commercially available (e.g., Amies’ or Stuarts’)**
**Specimens in transport media will remain viable for up to 4 days**
**Specimens should be labeled clearly (i.e. GBS or pen allergy)**

**CDC’s Recommendations for Prenatal Group B Strep Cultures ****(cont.)**

**Notes:**
CDC’S RECOMMENDATION FOR PRENATAL GROUP B STREP CULTURES (CONT.)
Prenatal specimen swabs should be put into nonnutritive transport medium (e.g., Amies’ or Stuarts’); in this medium, the specimens will remain viable for up to 4 days.
Specimens should be labeled to clearly identify it as a group B strep culture.  If susceptibility testing is required for clindamycin and erythromycin resistance because a patient is at high risk for penicillin anaphylaxis, the label should state this.  Laboratories and clinicians may come up with other strategies for implementing clindamycin and erythromycin susceptibility testing on prenatal GBS isolates of patients at high risk for penicillin anaphylaxis.

## CDC’s Recommendations for GBS Culture Processing

**Remove swabs from transport medium**
**Inoculate into selective medium**
**Commercially available brand (i.e. Lim Broth or Trans-Vag broth supplemented with 5% defribrinated sheep blood) **
**Incubate broth 18-24 h. Subculture to sheep blood agar plate**
**Inspect and identify suggestive organisms **
**B-hemolytic or nonhemolytic, Gram+, catalase-**

**Notes:**
LABORATORY ALGORITHM, CONTINUED
When swabs arrive at laboratory, remove swabs from transport medium
Inoculate into appropriate selective medium; such as a commercially available brand (i.e. Lim Broth or Trans-Vag broth supplemented with 5% defibrinated sheep blood)
Incubate broth 18-24 hours. Subculture to sheep blood agar plate.
Inspect and identify suggestive organisms: 
	B-hemolytic or nonhemolytic, Gram+, catalase-

## What is ‘Selective Broth Medium’?

**Todd-Hewitt broth supplemented with EITHER:**
**nalidixic acid (15 μg/ml) and colistin(10 μg/ml)**
**nalidixic acid (15 μg/ml) and gentamicin          (8 μg/ml)**
**Appropriate media are commercially available (e.g. Trans-Vag broth supplemented with 5% defribrinated sheep blood, Lim broth)**

**Notes:**
WHAT IS ‘SELECTIVE BROTH MEDIUM’?
An enriched liquid medium that encourages the growth of group B strep better than agar media
Supplemented with antibiotics to inhibit the growth of non-GBS organisms.
Food and Drug Administration (FDA) has issued statement recommending  use as standard means of identifying GBS from prenatal specimens.
Todd-Hewitt broth supplemented with EITHER:
	- nalidixic acid (15 μg/ml) and colistin (10 μg/ml)
	- nalidixic acid (15 μg/ml) and gentamicin (8 μg/ml)
Appropriate media are commercially available (e.g. SBM, Lim broth)

## CDC’s Recommendations for GBS Culture Laboratory Processes

**Various GBS antigen detection tests (i.e slide agglutination, genetic probes) may be used for specific identification or CAMP test for  presumptive identification**

**CDC’s Recommendations for GBS Culture Laboratory Processes**
**(cont.)**

**Notes:**
CDC’S RECOMMENDATIONS FOR GBS CULTURE LABORATORY PROCESSES
If GBS not identified after 18-24h on sheep blood agar plate, reincubate and inspect at 48h 
Various GBS antigen detection tests (i.e. slide agglutination, genetic probes) may be used for specific identification or CAMP test for  presumptive identification

## Procedure for Susceptibility Testing for Clindamycin and Erythromycin

**Use swab to make suspension from 18-24 hr growth of GBS in saline or Mueller-Hinton broth**
**Dip sterile swab in adjusted suspension**
**Inoculate entire surface of Mueller-Hinton sheep blood agar plate**
**Place clindamycin disk onto half of plate, erythromycin disk on other half **
**Incubate for 20-24 hrs**
**Measure diameter of zone of inhibition**
**Interpret according to NCCLS guidelines**

**Notes:**
PROCEDURE FOR SUSCEPTIBILITY TESTING FOR CLINDAMYCIN AND ERYTHROMYCIN
Use a cotton swab to make a suspension from 18-24 hr growth in saline or Meuller-Hinton broth to match a 0.5 McFarland turbidity standard
Within 15 minutes of adjusting turbidity, dip a sterile cotton into the adjusted suspension.
Use swab to inoculate the entire surface of a Mueller-Hinton sheep blood agar plate
After the plate dries, use sterile forceps to place a clindamycin (2 micrograms) disk onto half of plate and erythromycin (15 micrograms) disk onto the other half.
Incubate at 35C in 5% CO2 for 20-24 hours
Measure the diameter of the zone of inhibition using a ruler or calipers.
Interpret according to NCCLS guidelines for Streptococcus other than S. pneumoniae 
For detailed instructions, see Box 1 in the 2002 GBS prevention guidelines. They are found in the MMWR, Volume 51, RR-11

## Susceptibility Testing for Clindamycin and Erythromycin Resistance

**Increase in resistance to clindamycin and erythromycin**
**New intrapartum prophylaxis (IAP) regimens in 2002 guidelines for patients with penicillin allergy**
**Increased susceptibility testing may be ordered by health care providers **

**Notes:**
SUSCEPTIBILITY TESTING FOR CLINDAMYCIN AND ERYTHROMYCIN RESISTANCE
To date, there have been noticeable increases in GBS isolates resistant to clindamycin and erythromycin 
Because of this increase, new intrapartum prophylaxis regimens are included in the 2002 guidelines for women with penicillin allergy.
As a result, a greater number of susceptibility tests may be ordered by health care providers patients with penicillin allergy. Alternatively, some laboratories have chosen to institute a policy of testing susceptibility on all prenatal GBS specimens

## Susceptibility of GBS: ABC/EIP Isolates, 1995-2000

**1280 isolates from MN, GA, NY, OR (1173 invasive, 107 colonizing):**
**All susceptible to penicillin, ampicillin, cefotaxime and vancomycin**
**19% resistant to erythromycin  **
**11% resistant to clindamycin**

**Notes:**
SUSCEPTIBILITY OF GBS:  ABC/EIP ISOLATE, 1995-2000
1280 isolates from MN, GA, NY, OR (1173 invasive, 107 colonizing)
All susceptible to penicillin, ampicillin, cefotaxime and vancomycin
 19% resistant to erythromycin  
 11% resistant to clindamycin  

## Slide 17

**Notes:**
RECOMMENDED REGIMENS FOR INTRAPARTUM ANTIMICROBIAL PROPHYLAXIS FOR PERINATAL GBS DISEASE PREVENTION
Recommended:  Penicillin G, 5 million unites IV initial dose, then 2.5 million units IV every 4 hours until delivery
Alternative:  Ampicillin, 2 g IV initial dose, then 1 g IV every 4 hours until delivery
If penicillin-allergic:
Patients not at high risk for anaphylaxis:
Recommended – Cefazolin, 2g IV initial dose, then 1 g IV every 8 hours until delivery
Patients at high risk for anaphylaxis:
GBS susceptible to clindamycin and erythromycin – Clindamycin, 900 mg IV every 8 hours until delivery OR Erythromycin, 500 mg IV every 6 hours until delivery
GBS resistant to clindamycin or erythromycin or susceptibility unknown – Vancomycin, 1g IV every 12 hours until delivery

## Impact of new recs on clinical laboratories

- Many labs will receive more prenatal GBS cultures than they’re used to

- Even labs used to processing prenatal cultures will need a GBS susceptibility testing plan

**--****how to identify isolates for testing**

**--****how to conduct susceptibility testing**

**--how to communicate susceptibility results**

## What Can You Do to Help?

- Make sure your OB, Peds, and Microbiology colleagues know the new guidelines are out!
- Check if your lab is using selective broth medium.
- Form a committee to plan steps needed for implementation in your facility

## Laboratory-specific issues

- Assess personnel/supplies re: anticipated increase in GBS prenatal cultures
- Plan for how to report GBS from prenatal urine cultures (any concentration of GBS should be reported to provider)
- Susceptibility testing of GBS if Pen allergy
- Timely communication of results

## 2002 GBS Guidelines:

**Laboratory-Related Information **

**MMWR Vol. 51, RR-11**
**Box 1. Procedures for collecting and processing group B strep cultures and performing susceptibility testing to clindamycin and erythromycin (page 4)**
**Resistance in GBS (page 9)**
**Increased Incidence or Resistance in Non-GBS Pathogens (page 10)**
**Recommendations (page 16)**

**Notes:**
2002 GBS GUIDELINES:  LABORATORY-RELATED INFORMATION
The guidelines are located in MMWR Vol. 51, RR-11
Box 1. Procedures for collecting and processing group B strep cultures and performing susceptibility testing to clindamycin and erythromycin (page 4)
Resistance in GBS (page 9)
Increased Incidence or Resistance in Non-GBS Pathogens (page 10)
Recommendations (page 16)

## Key GBS Resources

- CDC's GBS Internet page
- http://www.cdc.gov/groupbstrep

**AAP endorsed CDC’s guidelines, ACOG and ACNM will release statements this winter, and AAFP is planning endorsement**

##                Close up of GBS Colony

-                Close up of GBS Colony

-   GBS Colony

- Photo courtesy of Dr. Ellen Jo Baron, Stanford, CA

**Notes:**
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

-     Comparison of GBS & GAS Hemolysis

- GAS

- GBS

- Photo courtesy of Dr. Richard Facklam, CDC

**Zone of hemolysis**

**Zone of hemolysis**

**Notes:**
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 Hemolytic Activity in stabbed portions of agar plate for  Group A,B,G, F Strep

- A

- F

- B

- G

- Photo courtesy of Dr. Richard Facklam, CDC

**Notes:**
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 Commercial Tests

-  Slides of  Agglutination Commercial Tests

-                  A  positive agglutination    A  negative agglutination
- 	      GBS is present                  GBS is not present

- Photo courtesy of Dr. Richard Facklam, CDC

**Notes:**
     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

-           Positive for GBS		    Not Positive for GBS

-  Positive zone of enhanced          hemolytic activity.

- Photo courtesy of Dr. Richard Facklam, CDC

- Negative reaction of non-enhanced zone of hemolytic activity.

**Notes:**
	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

-               Commercial Betalysin CAMP Test

** ****Zone of enhanced hemolytic activity**

- Photo courtesy of Dr. Richard Facklam, CDC

**Notes:**
	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 Test  (Test for Free Benzenze)

**   **** **

- Photo courtesy of Dr. Richard Facklam, CDC

- Positive for hippurate hemolysis

**Notes:**
	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 Test  (Rapid Hippurate Test)

- Photo courtesy of Dr. Richard Facklam, CDC

- Positive for hippurate hemolysis.

**Notes:**
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  E TestTM & Disk Diffusion 

- E TestTM

- Disk Diffusion

- Minimum inhibitory concentration

- Zone of inhibition of growth is larger than 26mm, meaning the bacteria is susceptible.

- Photo courtesy of Dr. Richard Facklam, CDC

**Notes:**
	 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 

-            Antimicrobial Susceptibility Test
- 		         Broth Dilution

- 		 (MIC) Minimum Inhibitory Concentration

-  MIC susceptible  for GBS

-  MIC  resistant  for GBS

- Photo courtesy of Dr. Richard Facklam, CDC

- Penicillin

- Ampicillin

- Erythromycin

**Notes:**
	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.