Sep 11, 2020
Isolation, Identification, and Antimicrobial Susceptibility Testing of Streptococcus pneumoniae
- Korrie Salsabila1,
- Wisnu Tafroji2,
- Wisiva Tofriska Paramaiswari1,
- Miftahuddin majid khoeri1,
- Dodi Safari1
- 1Eijkman Institute for Molecular Biology;
- 2Eijkman Institute for Molecular Biology, Jakarta, Indonesia
- Eijkman Institute for Molecular Biology
External link: https://doi.org/10.1371/journal.pone.0246122
Protocol Citation: Korrie Salsabila, Wisnu Tafroji, Wisiva Tofriska Paramaiswari, Miftahuddin majid khoeri, Dodi Safari 2020. Isolation, Identification, and Antimicrobial Susceptibility Testing of Streptococcus pneumoniae. protocols.io https://dx.doi.org/10.17504/protocols.io.bkuakwse
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 04, 2020
Last Modified: September 11, 2020
Protocol Integer ID: 41570
Keywords: Streptococcus pneumoniae, isolation, identification, antimicrobial,
Abstract
Streptococcus pneumoniae (S. pneumoniae) is a major cause of bacterial pneumonia and meningitis globally. This bacterium is a normal flora of nasopharynx but can move to sterile sites such as cerebrospinal fluid, blood, and pleural fluid then causes invasive pneumococcal diseases. However, antibiotics resistance causes the failure of pneumococcal disease treatment. S. pneumoniae penicillin-resistant is one of the priority antimicrobial resistant (AMR) pathogens in WHO list. Since pneumococcus is a fastidious bacteria, isolation and identification of S. pneumoniae is challenging. This bacteria need good quality of blood agar plate to grow and proper method to identify S. pneumoniae. Performing antimicrobila susceptibility testing of S. pneumoniae isolates is important to monitor and handle its resistance. Therefore, we decided to write the protocol regarding isolation, identification, and antimicrobial susceptibility testing of S. pneumoniae.
Materials
MATERIALS
TSA II Trypticase Soy Agar ModifiedBecton-DickinsonCatalog #212305
DD1 OptochinOxoid Microbiology Products - Thermo FischerCatalog #X3483A
Sodium deoxycholateSigmaCatalog #SRE0046-100G
Mueller-Hinton AgarOxoid Microbiology Products - Thermo FischerCatalog #CM0337
Tris-EDTA buffer solutionSigma – AldrichCatalog #933283
Skim MilkBecton-DickinsonCatalog #232100
Tryptone Soya BrothOxoid Microbiology Products - Thermo FischerCatalog #CM0129
DextroseBecton-DickinsonCatalog #215530
GlycerolSigmaCatalog #G5516
McFarland Equivalence Turbidity StandardCatalog #R20421
ClindamycinOxoid Microbiology Products - Thermo FischerCatalog #CT0064B
ErythromycinOxoid Microbiology Products - Thermo FischerCatalog #CT0020B
OxacillinOxoid Microbiology Products - Thermo FischerCatalog #CT0159B
ChloramphenicolOxoid Microbiology Products - Thermo FischerCatalog #CT0013B
TetracyclineOxoid Microbiology Products - Thermo FischerCatalog #CT0054B
Sulphametoxazole/TrimethoprimOxoid Microbiology Products - Thermo FischerCatalog #CT0052B
Gentamicin SolutionSigmaCatalog #G1397
FLOQ SwabCopanCatalog #503CS01
Nasopharyngeal Swab Collection
Nasopharyngeal Swab Collection
Nasopharyngeal swab collection follow method as described previously
CITATION
. A flexible nasopharyngeal flocked swab is slowly passed into nasopharynx. The swab should not be resistant before it was reached nasopharynx (NP). The nasopharynx distanced one-half to two-third from nostril to ear lobe
Once the swab reaches nasopharynx, it is rotated 3-5 times to let the mucus absorb into the swab
The swab is removed slowly and immadiately placed into 1 mL of Skim milk Tryptone Glucose and Glycerol (STGG). STGG 100 mL was made by mixing 2 g skim milk powder, 3 g Tryptone Soya Broth, 0.5 g glucose/dextrose, glycerol 10 mL and distilled water 90 mL
The NP-STGG is vortexed for 10-20 sec to disperse the bacteria from swab to the STGG then placed it in cryobox that filled by ice packs. The temperature should be4 °C and it is regularly monitored through the digital temperature controller
The inoculated STGG is kept at 4 °C for 4-6 hours after collection until reaches targeted laboratory. After that, the swab-STGG is frozen at -80 °C to secure survival of pneumococcus
The specimens are shipped to Molecular Bacteriology Unit, Eijkman Institute using dry ice.
Once it is arrived Eijkman Institute, the swabs were immadiately stored at -80 °C until culture.
Optochin Susceptibility Test
Optochin Susceptibility Test
NP-STGG medium that stroed in -80 °C is thawed and vortexed to disperse the bacteria from the swab.
20 µL of of swab-inoculated STGG was transferred and streaked onto blood agar plate (TSA II Trypticase Soy Agar Modified) with 8 % (v/v) sheep blood and supplemented with 5 mg/L gentamicin.
CITATION
The plate is incubated at 37 °C with 5% CO2 for 18-24 hours0 Mass Percent
Single colony of alpha-hemolytic and flat-depressed colony resembling pneumococci is picked and streaked onto blood agar plate (TSA II Trypticase Soy Agar Modified) with 8 % (v/v) sheep blood.
Quadran 1 and 2 is overlapped to test optochin susceptibility of the isolate. If there are 2 different colonies in the plate, each of them should be tested for optochin susceptibility separately.
Optochin disk (ethylhydrocupreine hydrochloride) is placed on the overlap area between quadran 1 and 2.
Susceptible isolate (zone ≥14 mm) with distinct characteristics of pneumococci (flat depressed center, shiny or wet colonies), is defined as Streptococcus pneumoniae (S. pneumoniae)
Overnight culture is harvested and put into STGG then stored in -80 °C
Bile Solubility Test
Bile Solubility Test
Resistant optochin isolate (zone ≤ 14 mm) which has characteristics of S. pneumoniae or susceptible isolate which has no pneumococcus disticnt characteristics (too dry colonies), is contined to bile solubility testing
Note
For each isolate tested, two glass tubes are prepared and labeled as ‘test tube’ and ‘control tube’
2 mL and 1 mL of saline 0.85% are added into test tube and control tube, respectively
Bacterial suspension that equvialent to MacFarland standard 1 is made in the test tube. Bacterial density is measued by densitometer (make sure to calibrate prior to using)
1 mL of bacterial suspension from test tube is transferred into control tube then vortexed.
1 mL of bile salt or sodium deoxycholate 2 Mass Percent is added into test tube. The final volume of test tube and control tube are 2 mL for each
ATCC 49619 of S. pneumoniae must be included in every bile testing
Test tube and control tube are incubated room temperature for 10 minutes. If the solution in the test tube of isolate is as clear as the test tube of ATCC 49619 (the soulution should be as clear as water), the isolate is bile-soluble and defined as S. pneumoniae. Otherwise, if there is no turbidity difference between test tube and control tube of the isolate, the isolate was not bile-soluble
If the isolate is negative for the first 10 minutes incubation, then continue for incubation at room temperature for 2 hours
Bile soluble isolate is streaked onto blood agar plate (TSA II Trypticase Soy Agar Modified) with 8 % (v/v) sheep blood and incubated at 37 °C with 5% CO2for 18-24 hours
Bile-soluble isolates were harvested and put into labeled STGG for freezing stock then vortexed. The bacteria should be well-hemogenized in STGG
STGG-inoculated isolate is stored in -80°C
DNA Extraction of S. pneumoniae
DNA Extraction of S. pneumoniae
The DNA extraction ofS. pneumoniae is performed by boil method as described previously
CITATION
Pure colonies ofS. pneumoniaestored in STGG at-80°C are streaked onto 8% sheep blood agar plate. Inoculated STGG must be kept at 4 °C
Incubation at 37 °C for 18-24 h with 5% CO2.
Single colony of the isolate is picked and streaked onto 8% sheep blood agar plate then incubated for 18-24 h at 37 °C with 5% CO2.
Fresh culture is harvested into TE buffer in 1.5 mL microcentrifuge tube and vortexed until homogenized
Bacterial suspension is heated at 100 °C for 00:05:00 and immadiately placed at freezer -20 °C 00:05:00
Centrifuge at 13000 x g, 00:10:00 then kept at -20 °C until serotyping
Serotyping of S. pneumoniae
Serotyping of S. pneumoniae
Serotyping ofS. pneumoniaewas performed by sequential multiplex conventional PCR targeted wzy gene and consisted of 8 reactions
CITATION
. Latin America scheme as described by Centers for Disease Control and Prevention, is followed for serotyping the isolates
Latin America Scheme Multiplex PCR.pdf . Every reaction should be detect cpsA gene. Each reaction has different serotypes to be identifiedPCR Oligonucleotide Primers.pdf .
Latin America Scheme Multiplex PCR.pdf . Every reaction should be detect cpsA gene. Each reaction has different serotypes to be identifiedPCR Oligonucleotide Primers.pdf .Note
Reaction 1: serotype14, 6A/6B/6C/6D, 23F, 19A, and 9V/9A.
Reaction 6C: serotype 6A/6B/6C/6D and 6C/6D.
Reaction 2: serotype 19F, 3, 15B/15C, 18C/18F/18B/18A, and 17F.
Reaction 3: serotype 1, 5, 9N/9L, 7F/7A, and 16F.
Reaction 4: serotype 8, 2, 4, 20, and 22F/22A.
Reaction 5: serotype 7C/7B/40, 12F/12A/44/46, 11A/11D, 10A, and 23A.
Reaction 6: serotype 21, 33F/33A/37, 15A/15F, 35F/47F, and 13.
Reaction 7: serotype 39, 23B, 35A/35C/42, 38/25F/25A and 35B.
Reaction 8: serotype 24F/24A/24B, 10F/10C/33C, 34, 19Fvar, and 31.
Mastermix for each reaction (25 μl) is prepared as described in Latin America scheme spreadsheet. For example, reaction 1 is performed by adding PCR H2O 0.5 µL of 5× PCR buffer (Promega), 3.5 µL of MgCl2 25 mM, 1 µL of dNTPs (Promega) 5 mM, varied volume for 25 μM forward primer and 25 μM reverse primer, 0.2 µL Taq Polymerase 2U and 2.5 µL of DNA template, into 1.5 mL of microsentrifuge tube.
For isolates serotyping, conventional PCR is run under following condition: pre-denaturation at 94 °C for 00:04:00 , followed by 30 cycles of denaturation at 94 °C for 00:00:45 , annealing at 54 °C for 00:00:45 , and extention at 65 °C for 00:02:30 .
The DNA is visualized through electrophoresis. Agarose 2 Mass Percent is made by adding 2 gram of agarose into 100 mL of Tris Acetat EDTA (TAE) 1× then heated in the microwave.
Agarose is poured into electrophoresis tank with the comb.
Agarose gel is placed into electrophoresis tank filled with cold TAE 1×
PCR product and DNA ladder 50 bp are added into each well of agarose gel
Voltage is set to 100V and run for 90 minutes
DNA bands are visualized using GelDoc machine
Isolate that serotype-negative is confirmed by real-time PCR targeting lytA to confirm that the isolate wasS. pneumoniae. For isolate with positive lytA is assigned as non-typeable (NT) by PCR S. pneumoniae
CITATION
Antimicrobial Susceptibility Testing
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing is performed by following Clinical Laboratory Standard Institutes (CLSI) 2017 guidelines
CITATION
. Frozen isolate in STGG was streaked onto 8 % (v/v) sheep blood agar plate. The STGG must be kept at 4 °C condition.
Single colony of pneumococci is picked and cultured onto 8% sheep blood agar plate then incubated for 18-24h at 37°C with 5% CO2.
Bacterial suspension is made by harvesting freshly grownS. pneumoniaeand put into 5 mL saline 0.85 Mass Percent
The suspension is adjusted to be equivalent as McFarland standard 0.5. Densitometer is used to measure the turbidity. Calibration using McFarland standards is done prior to measurement.
When the suspension reaches 0.5 McFarland standard, sterile cotton swab is dipped into bacterial suspension then pressed gently on the wall of glass tube to remove excess fluid.
The cotton swab is lawned confluently onto Mueller Hinton Agar (MHA) plate with 5 % (v/v) sheep blood and streaked 3 times by rotating in 60° direction.
Antibiotic disks are placed onto the agar and pressed gently by sterile pinset.
Note
Clindamycin 2 μg, chloramphenicol 30 μg, oxacillin 1 μg, tetracycline 30 μg, erythromycin 15 μg, and trimethophrim/sulfamethoxazole 1,25/23.75 μg, are used. Oxacillin disk is applied to measure susceptibility of isolates to penicillin.
Inoculated MHA is incubated at 37 °C with 5% CO2 for 20-24 h.
Inhibition zone is measured and recorded. Interpretation of clear zone follow Clinical Laboratory Standards Institute guideline 2017.
Citations
Step 1
Satzke C, Turner P, Virolainen-Julkunen A, Adrian PV, Antonio M, Hare KM, et al.. Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: Updated recommendations from the World Health Organization Pneumococcal Carriage Working Group
http://dx.doi.org/10.1016/j.vaccine.2013.08.062Step 28
Pai R, Gertz RE, Beall B. Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates
10.1128/JCM.44.1.124–131.2006Step 34
Carvalho M d. G, Pimenta FC, Jackson D, Roundtree A, Ahmad Y, Millar EV, et al.. Revisiting Pneumococcal Carriage by Use of Broth Enrichment and PCR Techniques for Enhanced Detection of Carriage and Serotypes
JOURNAL OF CLINICAL MICROBIOLOGY, May 2010, p. 1611–1618Step 43
Carvalho M d. GS, Tondella ML, McCaustland K, Weidlich L, McGee L, Mayer LW, et al. Evaluation and Improvement of Real-Time PCR Assays Targeting lytA, ply, and psaA Genes for Detection of Pneumococcal DNA
10.1128/JCM.02498-06Step 44
Clinical and Laboratory Standards Institute. M100 Performance standards for antimicrobial susceptibility testing 27th Edition (2017)
Step 9
Hadinegoro SR, Prayitno A, Khoeri MM, Djelantik IGG, Dewi NE, Indriyani SAK, et al.. Nasopharyngeal Carriage of Streptococcus pneumoniae in Healthy Children Under Five Years Old in Central Lombok Regency, Indonesia.