TITLE: Isolation, Identification, and Antimicrobial Susceptibility Testing of Streptococcus pneumoniae
AUTHORS: Korrie Salsabila,  Wisnu Tafroji,  Wisiva Tofriska Paramaiswari, Miftahuddin Majid Khoeri, Dodi Safari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Streptococcus pneumoniae</span><span> (</span><span style = "font-style:italic;">S. pneumoniae</span><span>) 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. </span><span style = "font-style:italic;">S. pneumoniae </span><span>penicillin-resistant is one of the priority antimicrobial resistant (AMR) pathogens in WHO list. Since pneumococcus is a fastidious bacteria, isolation and identification of </span><span style = "font-style:italic;">S. pneumoniae </span><span>is challenging. This bacteria need good quality of blood agar plate to grow and proper method to identify </span><span style = "font-style:italic;">S. pneumoniae</span><span>. Performing antimicrobila susceptibility testing of </span><span style = "font-style:italic;">S. pneumoniae</span><span> isolates is important to monitor and handle its resistance. Therefore, we decided to write the protocol regarding isolation, identification, and antimicrobial susceptibility testing of </span><span style = "font-style:italic;">S. pneumoniae</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. [Nasopharyngeal Swab Collection]
Nasopharyngeal swab collection follow method as described previously. 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
?. [Nasopharyngeal Swab Collection]
Once the swab reaches nasopharynx, it is rotated 3-5 times to let the mucus absorb into the swab
?. [Nasopharyngeal Swab Collection]
The swab is removed slowly and immadiately placed into 1 mL of Skim milk Tryptone Glucose and Glycerol (STGG). STGG  was made by mixing  skim milk powder,  Tryptone Soya Broth,  glucose/dextrose, glycerol   and distilled water
100 mL
2 g
3 g
0.5 g
10 mL
90 mL
?. [Nasopharyngeal Swab Collection]
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 be  and it is regularly monitored through the digital temperature controller
4 °C
?. [Nasopharyngeal Swab Collection]
The inoculated STGG is kept at  for 4-6 hours after collection until reaches targeted laboratory. After that, the swab-STGG is frozen at  to secure survival of pneumococcus
4 °C
-80 °C
?. [Nasopharyngeal Swab Collection]
The specimens are shipped to Molecular Bacteriology Unit, Eijkman Institute using dry ice.
?. [Nasopharyngeal Swab Collection]
Once it is arrived Eijkman Institute, the swabs were immadiately stored at   until culture.
-80 °C
?. [Optochin Susceptibility Test]
NP-STGG medium that stroed in   is thawed and vortexed to disperse the bacteria from the swab.
-80 °C
?. [Optochin Susceptibility Test]
of of swab-inoculated STGG was transferred and streaked onto blood agar plate (TSA II Trypticase Soy Agar Modified) with   sheep blood and supplemented with 5 mg/L  gentamicin.
20 µl
?. [Optochin Susceptibility Test]
The plate is incubated at   with 5% CO2 for 18-24 hours
37 °C
?. [Optochin Susceptibility Test]
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   sheep blood.
?. [Optochin Susceptibility Test]
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 Susceptibility Test]
Optochin disk (ethylhydrocupreine hydrochloride) is placed on the overlap area between quadran 1 and 2.
?. [Optochin Susceptibility Test]
Susceptible isolate (zone ≥14 mm) with distinct characteristics of pneumococci (flat depressed center, shiny or wet colonies), is defined as Streptococcus pneumoniae (S. pneumoniae)
?. [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
https://www.cdc.gov/meningitis/lab-manual/chpt08-id-characterization-streppneumo.pdf
?. [Bile Solubility Test]
For each isolate tested, two glass tubes are prepared and labeled as ‘test tube’ and ‘control tube’
?. [Bile Solubility Test]
2 mL and 1 mL of saline 0.85% are added into test tube and control tube, respectively
?. [Bile Solubility Test]
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)
?. [Bile Solubility Test]
1 mL of bacterial suspension from test tube is transferred into control tube then vortexed.
?. [Bile Solubility Test]
of bile salt or sodium deoxycholate   is added into test tube. The final volume of test tube and control tube are   for each
1 mL
2 mL
?. [Bile Solubility Test]
ATCC 49619 of S. pneumoniae must be included in every bile testing
?. [Bile Solubility Test]
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
?. [Bile Solubility Test]
If the isolate is negative for the first 10 minutes incubation, then continue for incubation at room temperature for 2 hours
?. [Bile Solubility Test]
Bile soluble isolate is streaked onto blood agar plate (TSA II Trypticase Soy Agar Modified) with   sheep blood and incubated at   with 5% CO2for 18-24 hours
37 °C
?. [Bile Solubility Test]
Bile-soluble isolates were harvested and put into labeled STGG for freezing stock then vortexed. The bacteria should be well-hemogenized in STGG
?. [Bile Solubility Test]
STGG-inoculated isolate is stored in -80°C
?. [Optochin Susceptibility Test]
Overnight culture is harvested and put into STGG then stored in
-80 °C
?. [DNA Extraction of S. pneumoniae]
The DNA extraction ofS. pneumoniae is performed by boil method as described previouslyPure colonies ofS. pneumoniaestored in STGG at-80°C are streaked onto 8% sheep blood agar plate. Inoculated STGG must be kept at
4 °C
?. [DNA Extraction of S. pneumoniae]
Incubation at   for 18-24 h with 5% CO2.
37 °C
?. [DNA Extraction of S. pneumoniae]
Single colony of the isolate is picked and streaked onto 8% sheep blood agar plate then incubated for 18-24 h at   with 5% CO2.
37 °C
?. [DNA Extraction of S. pneumoniae]
Fresh culture is harvested into TE buffer in   microcentrifuge tube and vortexed until homogenized
1.5 mL
?. [DNA Extraction of S. pneumoniae]
Bacterial suspension is heated at  for   and immadiately placed at freezer
100 °C
-20 °C
?. [DNA Extraction of S. pneumoniae]
Centrifuge at   then kept at   until serotyping
Centrifuge: 13000 34, 10 min
-20 °C
?. [Serotyping of S. pneumoniae]
Serotyping ofS. pneumoniaewas performed by sequential multiplex conventional PCR targeted wzy gene and consisted of 8 reactions. Latin America scheme as described by Centers for Disease Control and Prevention, is followed for serotyping the isolates. Every reaction should be detect cpsA gene. Each reaction has different serotypes to be identified.
Reaction 1: serotype14, 6A/6B/6C/6D, 23F, 19A, and 9V/9A.
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							Reaction 6C: serotype 6A/6B/6C/6D and 6C/6D.
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							Reaction 2: serotype 19F, 3, 15B/15C, 18C/18F/18B/18A, and 17F.
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							Reaction 3: serotype 1, 5, 9N/9L, 7F/7A, and 16F.
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							Reaction 4: serotype 8, 2, 4, 20, and 22F/22A.
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							Reaction 5: serotype 7C/7B/40, 12F/12A/44/46, 11A/11D, 10A, and 23A.
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							Reaction 6: serotype 21, 33F/33A/37, 15A/15F, 35F/47F, and 13.
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							Reaction 7: serotype 39, 23B, 35A/35C/42, 38/25F/25A and 35B.
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							Reaction 8: serotype 24F/24A/24B, 10F/10C/33C, 34, 19Fvar, and 31.
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?. [Serotyping of S. pneumoniae]
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  of 5× PCR buffer (Promega),   of MgCl2 25 mM,  of dNTPs (Promega) 5 mM, varied volume for 25 μM forward primer and 25 μM reverse primer,   Taq Polymerase 2U and   of DNA template, into 1.5 mL of microsentrifuge tube.
0.5 µl
3.5 µl
1 µl
0.2 µl
2.5 µl
?. [Serotyping of S. pneumoniae]
For isolates serotyping, conventional PCR is run under following condition: pre-denaturation at   for  , followed by 30 cycles of denaturation at   for  , annealing at   for  , and extention at   for  .
94 °C
94 °C
54 °C
65 °C
?. [Serotyping of S. pneumoniae]
The DNA is visualized through electrophoresis. Agarose   is made by adding 2 gram of agarose into 100 mL of Tris Acetat EDTA (TAE) 1× then heated in the microwave.
?. [Serotyping of S. pneumoniae]
Agarose is poured into electrophoresis tank with the comb.
?. [Serotyping of S. pneumoniae]
Agarose gel is placed into electrophoresis tank filled with cold TAE 1×
?. [Serotyping of S. pneumoniae]
PCR product and DNA ladder 50 bp are added into each well of agarose gel
?. [Serotyping of S. pneumoniae]
Voltage is set to 100V and run for 90 minutes
?. [Serotyping of S. pneumoniae]
DNA bands are visualized using GelDoc machine
?. [Antimicrobial Susceptibility Testing]
Antimicrobial susceptibility testing is performed by following Clinical Laboratory Standard Institutes (CLSI) 2017 guidelines. Frozen isolate in STGG was streaked onto   sheep blood agar plate. The STGG must be kept at  condition.
4 °C
?. [Antimicrobial Susceptibility Testing]
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.
?. [Antimicrobial Susceptibility Testing]
Bacterial suspension is made by harvesting freshly grownS. pneumoniaeand put into 5 mL saline
?. [Antimicrobial Susceptibility Testing]
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.
?. [Antimicrobial Susceptibility Testing]
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.
?. [Antimicrobial Susceptibility Testing]
The cotton swab is lawned confluently onto Mueller Hinton Agar (MHA) plate with   sheep blood and streaked 3 times by rotating in 60° direction.
?. [Antimicrobial Susceptibility Testing]
Antibiotic disks are placed onto the agar and pressed gently by sterile pinset.
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.
?. [Antimicrobial Susceptibility Testing]
Inhibition zone is measured and recorded. Interpretation of clear zone follow Clinical Laboratory Standards Institute guideline 2017.
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?. [Antimicrobial Susceptibility Testing]
Inoculated MHA is incubated at  with 5% CO2 for 20-24 h.
37 °C
?. [Serotyping of S. pneumoniae]
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