TITLE: Group B Streptococcus (Streptococcus agalactiae) Isolation, Identification and Antimicrobial Susceptibility Testing
AUTHORS: Wisnu Tafroji, Maria da Gloria Carvalho, Fabiana C Pimenta, Miftahuddin  majid khoeri, Dodi Safari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol provides a guideline for isolation, identification and antimicrobial susceptibility testing by microdilution of Group B Streptococcus (GBS) / </span><span style = "font-style:italic;">Streptococcus agalactiae. </span><span>The isolation is done by incubation of swab specimens in LIM broth enriched with 1.0 mL of rabbit serum, followed by selective media chromoagar. The identification is performed according to biochemistry characteristics by observing CAMP activity. Definitive grouping was done by latex agglutination (Remel cat no. R62031), according to the manufacturer’s instructions. The DNA was obtained using the fast extraction methodology for </span><span style = "font-style:italic;">Streptococcus</span><span>isolates and serotyping of GBS isolates was performed by conventional multiplex PCR. Antimicrobial susceptibility testing by broth microdilution was performed following the recommendations of Clinical and Laboratory Standards Institute minimum inhibitory concentration (MIC) breakpoints.</span></div></div>

[STEPS]
?. [Broth enrichment V/R swab culture for enhanced GBS growth]
Thaw the Vaginal/Rectal (V/R)-STGG specimens at room temperature (25oC) and vortex for approximately 10-20 seconds. Re-freeze the specimen (i.e., the STGG) as soon as possible and keep it cool in an ice bath during processing.Avoid multiple freeze-thaw cycles whenever possible. Transfer 200 µl of the V/R-STGG to 5 ml of LIM broth (BD-BBL cat# 296266) containing 1 ml of rabbit serum (Gibco cat#16120099).Vortex and incubate the inoculated LIM broth for 5 hours at 37oC with 5% CO2.Vortex and inoculate one loop (10 µl) of the LIM broth enriched culture on CHROMID®Strepto B (BioMérieux cat#419751) and incubate for 18-24 hours at 37 oC in CO2-incubator.
?. [GBS isolates detection and identification]
Carefully examine the CHROMID®Strepto B, for typical GBS colonies, pale pink to red after 24 hours incubation.A single colony (suspected pink colony) should be passed to fresh blood agar plate (BAP) and incubate for 18-24 hours at 37oC in CO2-incubator. The GBS colonies are gray to whitish gray surrounded by a weak zone of beta hemolysis. Non-hemolytic isolates can also be encountered.The suspected GBS isolates should be submitted to identification test, including Gram stain, CAMP test and latex agglutination (PathDx Strep B latex – R62031).If confirmed identification all GBS isolates should be frozen from a fresh culture (24h) in STGG or sheep blood and kept at -70°C for further testing.
?. [To perform the CAMP-test]
The CAMP test (Christie-Atkins-Munch-Peterson) is a test to identify GBS based on their formation of a substance (CAMP factor) that enhanced the area of hemolysis formed by the beta-hemolysin elaborated from Staphylococcus aureus (ATCC 25923).The suspected GBS strain to be tested and the S. aureus (ATCC 25923) are streaked onto a BAP at 90o angle.Incubate at 37°C/CO2 for 18-24 hours. A positive reaction can be detected by the presence of a triangular zone of enhanced beta-hemolysis in the diffusion zone of S. aureus beta-hemolysin and the CAMP factor.Reference: Carrol, KC, Pfaller MA, Landry, ML, Patel, R, McAdam, AJ, Richter, SS. In Streptococcus, 399-417. Manual of Clinical Microbiology. 12th ed. Washington DC: American Society for Microbiology Press; 2019.
?. [To perform latex agglutination]
The latex agglutination is performed using PathDx Strep B latex – R62031.Clean and label the slide. Prepare the bacterial suspension by placing a drop of 0.85% saline on the slide.Harvest a heavy loop of fresh growth from BAP and mix with the saline on the slide in circular movements enough to make a milky suspension.Homogenize/shake the PathDx Strep B flask, place one drop of the latex on top of the milky bacteria suspension and rock the slide for 1 minute. If agglutination occurs, the culture is positive. Reference: According to the manufacturer’s instructions
?. [Storage of GBS isolates]
After confirmed identification all GBS isolates should be frozen from a fresh culture (24 hours) in STGG or sheep blood and kept at -70°C for further testing (serotyping, antimicrobial susceptibility, etc…).
?. [DNA fast extraction of Streptococcus]
1. In a BSC, prepare the initial bacteria suspension. Depending on the organism being extracted, use the following     amounts: For Streptococcus agalactiae, using a 1µl loop, transfer 2-3 colonies of overnight blood agar plate growth to a labeled microcentrifuge tube containing 300µl of 0.85% NaCl and vortex. 2. Heat the suspension at 70⁰C for 15 minutes, use cap holder to secure the lid.3. Spin microcentrifuge tube for 2 minutes in centrifuge at 10,000 rpm and remove supernatant.4. Re-suspend the pellet in 50µl TE buffer, add 12µl mutanolysin (solution 2500U/ml) and 8µl of hyaluronidase (solution 30mg/ml) and vortex.The mutanolysin solution is prepared by dissolving 10,000 units of mutanolysin (Sigma #M9901) into 4ml of PCR water (2500unit/ml stock solution); stored in aliquots of 500µl at -20⁰C. The hyaluronidase solution is prepared by dissolving 100mg hyaluronidase (Sigma #H3506) into 3.3ml PCR water (30mg/ml stock solution); stored in 500µl aliquots at -20⁰C. 2. Heat the suspension at 70⁰C for 15 minutes, use cap holder to secure the lid.3. Spin microcentrifuge tube for 2 minutes in centrifuge at 10,000 rpm and remove supernatant.4. Re-suspend the pellet in 50µl TE buffer, add 12µl mutanolysin (solution 2500U/ml) and 8µl of hyaluronidase (solution 30mg/ml) and vortex.The mutanolysin solution is prepared by dissolving 10,000 units of mutanolysin (Sigma #M9901) into 4ml of PCR water (2500unit/ml stock solution); stored in aliquots of 500µl at -20⁰C. The hyaluronidase solution is prepared by dissolving 100mg hyaluronidase (Sigma #H3506) into 3.3ml PCR water (30mg/ml stock solution); stored in 500µl aliquots at -20⁰C. 5. Heat the suspension in water bath at 37⁰C for 30 minutes to overnight.6. Heat the suspension to inactivate for 10 minutes in 100⁰C heat block, use cap holder to secure the lid.7. Spin the tube for 4 minutes in centrifuge, identify the tube with the cryogenic label and use the DNA template in the supernatant.8. Proceed immediately with PCR reaction or store lysates at -20⁰C until use.
?. [GBS serotyping by Conventional multiplex PCR]
A. Mastermix preparation and PCR set upPipet 4.95µl PCR grade water, 12.5µl 2X Buffer-Qiagen Multiplex PCR Kit (catalogue number 206143) and 1uM of each primer per reaction in the labeled microcentrifuge PCR master mix tube (multiply the volumes by the number of reactions).Vortex and spin briefly the tube with the prepared PCR master mix.Pipet 22.5µl PCR master mix in each labelled 96-Well Plate/strip well. Add 2.5µl PCR grade water to the well labeled as negative control. Cover the wells containing the PCR master mix with 8-cap strips and take the 96-Well Plate to another biosafety cabinet, if possible, in another room.Add 2.5µl of each DNA sample and S. agalactiae serotype DNA controls to the respective wells. Close the filled wells and press the caps tightly.Enter the amplification parameters on the thermal cycler as follows: 6. Check the sample volume and review the parameters on the thermo cycler screen and start the run.
?. [GBS serotyping by Conventional multiplex PCR]
2. Agarose gel preparationOn a precision scale, weigh 1.6g Nusieve® agarose (catalogue number 50090).Mix the weighed agarose with 80ml room temperature 1X TAE buffer (2% gel final concentration).Heat in microwave for 90 seconds. Using thermoresistant gloves, carefully mix the solution, checking against the light to see if the agarose is completely dissolved, adding more time in the microwave as needed.Let the agarose solution cool down to 50°C (about 5 minutes), add 5µl (0.5µg/µl) of EtBr.Pour the gel on a casting tray with a 24 wells comb and wait until it solidifies. If not using the gel in the same day, cover the gel with 1X TAE buffer and put it in the refrigerator until ready to use.
?. [GBS serotyping by Conventional multiplex PCR]
3. Gel loading and runningCarefully remove the comb and place the gel in the electrophoresis unity. Fill the unity with refrigerated 1X TAE buffer until it covers the gel completely.Load 15µl ready to use 50-bp ladder (catalogue number 70538-3) on the first and last gel wells, following the template.Mix 10µl amplified DNA with 1µl gel loading dye and load each, following the prearranged order on the template, in the gel wells. Set the voltage source to 100V for 90 minutes and turn electrophoresis unity on.
?. [GBS serotyping by Conventional multiplex PCR]
4. Gel picture and analysisGel picture:When the run is finished, turn the electrophoresis unity off, remove the gel and take it to a UV source to take a picture.The results on the gel picture should be compared to the S. agalactiae controls bands and the serotypes determined for the samples’ band sizes, that must exactly match the controls’ band sizes before assigning a serotype.When the run is finished, turn the electrophoresis unity off, remove the gel and take it to a UV source to take a picture.The results on the gel picture should be compared to the S. agalactiae controls bands and the serotypes determined for the samples’ band sizes, that must exactly match the controls’ band sizes before assigning a serotype.        Note:         Non-specific bands have been occasionally detected when performing multiplex PCR testing on clinical specimens.  3. Gel Picture Example:
?. [GBS serotyping by Conventional multiplex PCR]
5. Interpretation of ResultsSerotype results are defined by the presence of specific bands and band sizes shown by gel electrophoresis of each sample. Each sample must contain the 688 bp band (capsular gene cpsi) common to all serotypes.	 Serotype specific band sizes are shown in the table below to determine serotype based on band sizes seen for    sample.
?. [To perform antimicrobial susceptibility testing]
Obtain BAP with overnight growth of isolate to be tested. Examine culture for purity. It should contain unique colony type with the expected S. agalactiae morphology. The test should be performed only with pure culture.Using a sterile swab or a loop, transfer few colonies to 5 ml of saline or Mueller-Hinton broth. Vortex the suspension.Use the 0.5 McFarland standard and/or densitometer to determine turbidity of suspension. Add cells to obtain the 0.5 McFarland standard (1.5 x 108 CFU/ml) suspension. The suspension should be used within 15 minutes.Transfer 100 µl of the adjusted suspension to Mueller-Hinton broth with lysed horse blood. Mix the suspension gently by inversion and dispense into a sterile reservoir.Open the S. agalactiae MIC panel package with a disinfected scissor; label the panel with isolate ID and transfer 100 µl of the inoculated Mueller-Hinton broth with lysed horse blood broth to each well using a multichannel pipette. After finish the whole panel inoculation cover the top of the panel with a plate seal to avoid evaporation. Note: Make sure every well is covered or evaporation can occur.Incubate the inoculated MIC panel in ambient air at 35-37oC for 20-24 hours. When the incubation period is complete read the MIC panel with the help of parabolic magnifying mirror and light. Record the antimicrobial endpoint on the work sheet as the first well showing no readable visible growth. The endpoint is the first well showing no readable visible growth (100% inhibition) for that antimicrobial. The MIC results should be interpretive according to latest CLSI recommendations.Reference: Clinical and Laboratory Standards Institute. CLSI Document M100-S22. edn. Wayne, PA: Clinical and Laboratory Standards Institute; 2020.