Journal of Microbiology & Experimentation

Research Article Open Access

Isolation of Streptococcus pneumoniae from the

sputum samples and their antimicrobial resistance in
Biratnagar, Nepal
Abstract Volume 7 Issue 6 - 2019

Sputum is one of the best specimens for the isolation of respiratory tract infections
Sanjay Mahato,1,2 Hema Kumari Sah,2 Sudhir
pathogen. Ten sputum specimens were collected from different hospitals of Biratnagar city
where patients of age group 40–73 years suffering from headache, coughing, mild fever,
Yadav2
AASRA Research and Education Academy Counsel, Nepal
1
chest pain had visited. In this study, a pathogen of pneumococcal disease was isolated from
Department of Microbiology, Mahendra Morang Adarsha
2
the sputum sample in 5% goat blood agar by quadrant streak method. The predominant Multiple Campus, Tribhuvan University, Nepal
bacterial pathogen isolated from sputum sample was 10% Streptococcus pneumoniae and
90% S. viridian. An old patient of 73 years of age was identified positive with pneumococcal Correspondence: Sanjay Mahato, AASRA Research and
disease with organisms like S. pneumoniae and S. viridian. These pathogens were identified Education Academy Counsel, Biratnagar-6, Nepal, Tel +977-
by various biochemical test likewise, catalase, optochin, and bile solubility. S. pneumoniae 9865658585, Email
isolate exhibits high sensitivity against chloramphenicol and amoxicillin but resistance
against cefotaxime and erythromycin. Received: September 27, 2019 | Published: November 26,
2019
Keywords: sputum, Streptococcus pneumoniae, S. viridian, Biratnagar, resistance

Introduction of bacteria with physiologic and genetic properties suitable for

colonization and multiplication under certain conditions.8 The
Streptococcus pneumoniae (Previously Pneumococcus or spectrum of pneumococcal disease differs in different age groups and
Diplococcus pneumoniae) may occur intracellular or extracellular as different populations. Several risk factor for pneumococcal infection,
gram-positive lanceolate diplococci but can also occur as single cocci such as age, race, immunodeficiency, other illness, socio-economic
or in short-chain of cocci. S. pneumoniae is a fastidious bacterium, status, previous antibiotic therapy, and day-care attendance have been
which grows best at 35-37°C with ⁓5% CO2.1 It is generally cultured reported.9 The serious pneumococcal infections such as pneumonia,
on media that contain blood but can also be grown on a chocolate bacteremia, meningitis, and otitis media remain major causes of
plate agar. On blood agar plate, colonies of S. pneumoniae appear morbidity and mortality in persons of all age’s worldwide.10 Capsules
as small, grey, moist (sometime mucoidal), and typically producing contribute to the ability of pneumococci to cause disease by providing
a zone of α-hemolysis (green in color).1 The α-hemolytic property resistance to phagocytosis and promoting evasion of the host immune
differentiates this organism from many species, but not from system by the bacteria.11
viridians streptococci. It is difficult as young pneumococcal colonies
appear raised similar to viridians streptococci, however, once the Adult mortality rates for bacteremic pneumococcal pneumonia
pneumococcal culture ages 24-48 hours, the colonies become range from 10% to 30% and for meningitis from 16% to 37%.
flattened, and not occur with viridians streptococci.2 In a gram-stain, Mortality rates are substantially higher in the elderly and in patients
Pneumococcus appears as oval-shaped, gram-positive cocci, 1-2µm with comorbidities.12,13 Importantly, mortality rates are much higher
in diameter, typically in pairs, sometimes singly or short chains.3 It is in young children in developing countries 10% to 40%, likely due
catalase-negative and facultative anaerobic but can grow aerobically. to poorer access to health care and comorbidities, particularly
Thus, it is recommended that cultures be incubated in a CO2 enriched HIV infection and malnutrition.14,15 A recent study of invasive
atmosphere.4 Susceptibility to optochin (ethylhydrocuprine) and bile pneumococcal disease (IPD) cases in Denmark confirmed that age
solubility are used to differentiate Streptococcus pneumoniae from and comorbidities influence survival. Mortality rates were ˂3% for
the other viridians streptococci. Optochin is an antibacterial agent not children younger than 5 years of age, 14% for individuals aged 5-65
used in therapy but only for the differentiation of Streptococcus.5 years of age, 24% for adult ≥65 years of age.16

Bacterial colonization begins immediately after birth and continues The emergence of drug-resistant Streptococcus pneumoniae will
throughout life with minor changes. Normal nasopharyngeal bacterial make these common infections more difficult to treat, as isolates
flora develops during the first year of life and the number of bacterial resistant to one or more first-line agents are common in many parts
species varies much.6 Normal flora plays a crucial role in the of the world.17 Since the first report of decreased susceptibility to
prevention of infectious diseases. However, a major part of infections penicillin in 1967, resistance of Streptococcus pneumoniae to this
is caused by microbes likes S. pneumoniae, H. influenza, N. meningitis drug, as well as other antibiotics, has been spread from country to
and Staphylococcus aureus, which originally belong to normal flora.7 country.18 Pneumococcal vaccines have been developed to prevent
pneumococcal infections. Susceptibility to the macrolides was
Infections caused by S. pneumoniae can be divided into two reduced among penicillin-resistant isolates.19 The lack of study and
categories mucosal infection such as otitis media and sinusitis and information in public domain had been a big reason for conducting
invasive infection such as septicemia, meningitis, and pneumonia. It this study regarding increasing pneumococcal resistance to commonly
belongs to the normal nasopharyngeal microbial flora that consists used antibiotics and its prevalence.

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©2019 Mahato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and build upon your work non-commercially.
Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Copyright:
©2019 Mahato et al. 300
Biratnagar, Nepal

Methodology grown for 18-24 hours on a blood agar plate at 37°C with approximately
5% CO2 (or in a candle-jar).1 Gram staining was performed on
Sample collection/research design the selected colonies. Gram-positive cocci bacteria with linear or
This cross-sectional observational study was conducted in the irregular arrangement was considered as possible streptococci. A
eastern region of Nepal at three centers of Biratnagar city (Nepal) negative catalase test further confirmed the isolate to be streptococci.
namely Koshi Zonal Hospital, Golden Hospital and Nepal Anti For catalase test, a pinch of bacterial colony was taken with the help
Tuberculosis Association (NATA) for duration of ten months (March of a sterile disposable loop and was mixed in 3% H2O2 to the grease
2017 to December 2017). The informed consent was taken from free slide. Immediate effervescence of bubble on the slide indicates
the patients. The study was approved by Mahendra Morang Adarsh positive test for catalase.22
Multiple Campus (MMAMC), Biratnagar and was technically Optochin test was done to confirm streptococci.1,22 The strain to
supported by AASRA Research and Education Academy Counsel, be tested was grown on a blood agar plate (BAP) at 37°C for 18-
Biratnagar. The study was carried out in the Microbiology Laboratory 24 hours with ⁓5% СО2 (or in a candle-jar). A disposable loop was
of MMAMC, Biratnagar, Nepal. A total of ten patients were tested used to remove an isolated colony from the overnight culture on the
during the study period. Demographic profile like age, gender and BAP and streak on to one half of a BAP. An optochin disc (5µg) was
symptom of the patients was taken. The mouth of patient was rinsed placed within the streaked area of the BAP and incubated overnight
with water. Deep breath was taken through mouth and coughed up at 37°C with ⁓5% CO2. The growth on the BAP near the optochin
mucous with deep coughing. To avoid contamination, each patient/ disc was observed and the zone of inhibition was measured. A zone
individual was instructed on how to collect a cough specimen by of inhibition of 14 mm or greater was indicated sensitive and was
laboratory personnel. The specimen was collected in a 20mL sterile considered for presumptive identification of pneumococcus.
screw-capped and wide-mouthed universal container. The container
with the specimen was appropriately labeled with a unique sample Bile solubility test: Ten grams of sodium deoxycholate was dissolved
number, date, and time of collection. After collection, it was into 100 ml sterile distilled water.1,22 A loopful of 10% deoxycholate
forwarded to the Microbiology laboratory of MMAMC for culture solution was placed on overnight grown prospective S. pneumoniae
and drug susceptibility testing. The specimen was analyzed within 2 colony on BAP. The colony lysed within a few minutes confirmed the
hours after collection. isolate as S. pneumoniae.

Whole goat blood collection and defibrination Antibiotic susceptibility study

Infection free goat was selected for blood collection.20 The For antimicrobial testing of streptococci, 5% defibrinated sterile
goat head was in halter position so that it was slightly elevated and goat blood was aseptically mixed to molten Mueller-Hinton Agar before
drawn from the side opposite to jugular vein. The vein puncture area plating. Antibiotic susceptibility testing of streptococci was performed
(approximately 100cm2) was disinfected with 70% alcohol. The vein by disc diffusion method (Kirby-Bauer method) on Muller-Hinton
was occulated by applying hand pressure in the juglar groove located agar (Himedia, Mumbai, India) supplemented with 5% defibrinated
in the lower neck. Goat blood is obtained by venipuncture via the sterile blood and was interpreted according to the Clinical Laboratory
jugular vein. The blood was withdrawn and collected in sterile conical Standard Institute (CLSI) guidelines.23 A homogenous suspension of
flask containing sterile glass beads. Defibrination of whole goat blood 0.5 MacFarland standard of a pure colony (not more than 48 hours)
was done using glass beads.21 The glass beads were soaked in 1M HCl was prepared in 5 mL of sterile normal saline (0.85% NaCl). Using
for overnight. Nearly 10-20 g of the glass beads of 4 mm size was a sterile swab, the bacterial suspension was evenly distributed over
placed in the 250 ml of conical flask and was sterilized by autoclaving. the entire surface of blood supplemented MHA plates. The antibiotic
100 ml of goat blood was measured and added aseptically to the flask disc (Himedia, Mumbai, India) containing the following antibiotics
of beads/tiles and sealed tightly. The conical flask was shaken gently. was used: Amoxicillin (AMX, 10μg), Cefotaxime (CTX, 30μg),
When the process was completed, the sound of the beads in a conical Chloramphenicol (C, 30μg), and Erythromycin (ERY, 15μg). Once
flask was changed from clear to dull. The defibrinated whole blood the discs were applied onto the plates, the plates were incubated at 37
was aspirated aseptically and separated. °C for 24 hr. Zone of inhibition was measured and interpreted using
the standard chart and the organisms were reported as susceptible,
Sample processing and isolation of microbes intermediate, or resistant accordingly.
For the preparation of 5% blood agar, 7.4 g of blood agar base Result
powder (Himedia, Mumbai, India) was added in 95 ml of distilled
water and was sterilized by autoclaving.1 The media was cooled to The results were obtained from sputum of more than 40 years
45°C keeping care that it didn’t start solidifying. Five milliliters of old patients of Koshi Zonal Hospital, Golden Hospital, and NATA at
the defibrinated blood was added aseptically in the blood agar base Biratnagar who was admitted for a week and were suffering from fever,
with constant shaking in such a way that forth formation prevented. headache, coughing, and chest pain. When ten samples were cultured
The blood agar was poured in sterile Petri-plates and was solidified. on 5% blood agar, only one sample (H1) showed α- hemolytic colonies
After solidification of media, specimen inoculated by quadrant streak of S. pneumoniae and S. viridans; while γ- hemolytic colonies were
method. Then plates were incubated in candle jar at 37°C for 18-24 seen in rest of the nine sample. None of the samples had β-hemolytic
hours. After incubation, all the plates were observed. S. pneumoniae colonies. Out of 10 samples, 10% (n=1) was α-hemolytic and 90%
and viridans streptococci showed α- hemolytic in 5% blood agar. (n=9) were γ-hemolytic streptococci. In blood agar, α-hemolysis
showed greenish coloration around the colonies; while in γ- hemolysis
Conventional identification of S. pneumococcus no zone was observed (Figure 1 & 2).
Gram staining and catalase test were performed on the isolates These isolated colonies were identified by conventional

Citation: Mahato S, Sah HK,Yadav S. Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Biratnagar, Nepal. J
Microbiol Exp. 2019;7(6):299‒304. DOI: 10.15406/jmen.2019.07.00274
Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Copyright:
©2019 Mahato et al. 301
Biratnagar, Nepal

identification methods like Gram staining, catalase test, optochin Antibiotic susceptibility test showed that S. pneumoniae was
sensitivity test, and bile solubility test. Colonies which were round, sensitive to amoxicillin and chloramphenicol, but, was resistant to
white grey with smooth margin, 1 mm in diameter, and 0.5 mm zone cefotaxime and erythromycin (Figure 5 & 6).
of α-hemolysis showed optochin sensitivity and bile solubility (Figure
3 & 4). This concluded the colonies were S. pneumoniae. Some other
specific colonies which were round, white grey, but, with rough
margin, having 1.5 mm size, and 1-2 mm zone of α-hemolytic didn’t
show optochin sensitivity and bile solubility. Thus, S. viridans was
identified.

Figure 4 Bile solubility test on blood agar.

Figure 1 α-hemolytic colonies of S. pneumoniae.

Figure 5 Antibiotic susceptibility test (AST) of S. pneumoniae.

Figure 2 γ-hemolytic colonies streptococci.

Figure 3 Optochin sensitivity on S. pneumoniae. Figure 6 AST of S. pneumoniae.

Citation: Mahato S, Sah HK,Yadav S. Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Biratnagar, Nepal. J
Microbiol Exp. 2019;7(6):299‒304. DOI: 10.15406/jmen.2019.07.00274
Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Copyright:
©2019 Mahato et al. 302
Biratnagar, Nepal

Table 1 Colony character and biochemical tests of S. pneumoniae and S. viridian in blood agar

α-hemolytic colonies
Blood agar/
Biochemical tests
S. pneumoniae S. viridans

Round initially, latter no change

Shape Round initially, latter draughtman’s colony.
occurs in colony.

Size 1mm 1.5mm

Zone of α-haemolysis 0.5mm 1-2mm

Margin Smooth Rough

Opacity Opaque Opaque

Colour White-grey colony White-grey colony

Gram stain +ve (lanceolate cocci) +ve (cocci)

Catalase -ve -ve

Optochin sensitivity Sensitive Resistant

Bile solubility +ve -ve

Discussion The antimicrobial sensitivity test of S. pneumoniae isolated from

H1 sample showed resistance towards cefotaxime and erythromycin;
When specimen was cultured on 5% blood agar, α-hemolytic while susceptibility to chloramphenicol and amoxicillin. Karcic
colonies were observed. Out of 10 samples, 10% (n=1) α-hemolytic et al had reported 45% of resistance to erythromycin and 0.56%
and 90% (n=9) γ-hemolytic streptococci were found. The findings of resistance to chloramphenicol.31 The resistance to cefotaxime in
of this study were slightly deviated from the study of Choi et al.,24 the present study contradicted the findings of Chawla et al which
reporting 5.9% viridans streptococci and 21.3% S. pneumoniae. showed 100% sensitivity to cefotaxime.32 Currently the antibiotic
α-hemolysis showed greenish coloration around the colonies in blood resistances pattern of S. pneumoniae isolate vary widely from
agar. It occurred due to partial lysis of red blood cell present in blood one country to another within Asia. China, India, and Philippines’
agar. In γ- hemolysis, there was no lysis of red blood cells and, hence. rate of erythromycin resistance is very high.33,34 Dias and Canica
There was no zone of lysis.1 Streptococcus pneumoniae showed demonstrated a clear correlation between erythromycin resistance and
catalase negative test because they didn’t produce catalase enzyme to high overall macrolide consumption, or conversely.35
break H2O2 into H2O and O2.25
The increased rate of erythromycin resistance can possibly be
The identified isolate Streptococcus pneumoniae was gram- correlated with the wide use of this antibiotic in the communities
positive lanceolate cell because of the binding of crystal violet to because of its dose convenience, cost-effectiveness, and easy
their cell structure. The cell wall of gram-positive organisms is more availability over the counter.36 Although multiple factors influence
sensitive to dehydration by alcohol, a substance that acts to close up the emergence and spread of antimicrobial resistance, random
the normal pores of the wall as it contains thick layer of peptidoglycan antimicrobial consumption is one of the most important.37 The
and very low concentration of lipid. Under these conditions, the large remarkable difference might have occurred because of the random
complexes of crystal violet iodine cannot escape from the cells, and use of antibiotics, the lack of antibiotic policy, and control in such
thus the primary color is retained by the gram-positive bacteria.26 countries.38
The α-hemolytic isolate was optochin sensitive and efficacy was
accompanied by anaerobic condition.27 Optochin caused fragility of
Conclusion
the bacterial cell membrane and lysed the bacterial cell due to changes The study concludes that α-haemolytic Streptococcus pneumoniae
in surface tension. In case of S. viridians, the optochin didn’t change shows resistance to erythromycin, cefotaxime while remains sensitive
the surface tension and, hence, the cell didn’t lyse.28 In bile solubility to amoxicillin and chloramphenicol. Chloramphenicol and amoxicillin
test, bile salt solution rapidly lysed pneumococcal colonies. Lysis can be an effective drug for the treatment of respiratory tract infection
depends on the presence of an autolytic amidase that cleaves the patients.
bond between alanine and muramic acid in the peptidoglycan of cell
wall.29 Bile salts lower the surface tension between the bacterial cell Acknowledgments
membrane and the medium; consequently, accelerating the organism’s
None.
natural autolytic process. Bile salt activates the autolytic enzyme
which induces flattening of the colonies.30

Citation: Mahato S, Sah HK,Yadav S. Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Biratnagar, Nepal. J
Microbiol Exp. 2019;7(6):299‒304. DOI: 10.15406/jmen.2019.07.00274
Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Copyright:
©2019 Mahato et al. 303
Biratnagar, Nepal

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Citation: Mahato S, Sah HK,Yadav S. Isolation of Streptococcus pneumoniae from the sputum samples and their antimicrobial resistance in Biratnagar, Nepal. J
Microbiol Exp. 2019;7(6):299‒304. DOI: 10.15406/jmen.2019.07.00274