Use of Genetic Methods for Detection of Streptococcus Pneumoniae Virulence Factors Isolated from Patient with Pneumonia
Sanaa Sauod Ahmed [email protected]
Department of Microbiology , College of Veterinary Medicine, University of Tikrit, Tikrit, Iraq Abstract
This study aimed to detection of Streptococcus pneumoniae and detection of their virulence factors , For this purpose 60 sputum samples were collected from patients with bacterial pneumonia all patient arrived to critical care unit (CCU). Bacterial culture technique and genetic technique were applied. The result showed that Streptococcus pneumonia detected in rate of 10% . results of mPCR showed that the PlyA&LytAdetected in rate of 100% while PsaAdetected in rate of 71.4%.
Pneumonia is an inflammation of lung parenchyma tissue. it caused by many bacterial spp. Such as Streptococcus pneumoniae , Haemophilusinfluenzae Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Moraxella catarrhalis, Neisseria meningitides (Welp&Bomberger, 2020)
Streptococcus pneumoniaebelongs to the family streptococcaceae. It is a gram-positive cocci , fastidious bacteria and facultative anaerobic, but can grow aerobically, caused α-hemolytic, and sensitive to optochin test. (Weisbroth, & Kohn, 2020)
Streptococcus pneumoniaeis one of normal nasopharyngeal microbial but may be became pathogen and caused many infection such as septicemia, meningitis, pneumonia, and mild upper respiratory infections, it can transmission by respiratory secretions that contaminated hand, instruments or airborne droplets (Aykacet al.,2020).
The virulence genes can be acquired by many way such as spontaneous mutations and gene exchange via horizontal gene transfer (D'Mello, 2021). And may be regulate the physical properties ( adhesion, biofilm, fimbriae, and flagella or regulation of biochemical factors (enzymes, toxins or antibiotics ) (Schulze et al.,2021)
Materials and methods
Samples: 60 sputum samples collected from patients with bacterial pneumonia (according to physician examination, chest X-Ray and Gram stain of sputum), all patient arrived to critical care unit (CCU) in Salahaldeen teaching hospital.
Bacterial culture : all sample cultured on Blood agar, Chocolate agar manitol salt agar, macConkey agar.
DNA extraction: DNA direct extraction from sputum by use of (Sputum DNA Isolation Kit – Morgen company -Product #: RU46100) and according to manufacturer’s instructions.
Primers used for of detection of S.pneumoniae : as in table (1).
Table (1): Primer used for detection of S.pneumoniae
Gene name Sequences Out product size References
F 5’-GAAGAGCCAAGGACAGGTAC-3’ 268 Jourdain et
- PCR Reaction mixture and thermocyclar program used for detection of S.pneumoniaeas in table (2) and table (3).
Table (2) PCR Reaction mixture for detection of S.pneumoniae
Master mix components Amount (μM)
Master Mix 20
Forward prime (Beta-globin) 1
Reverse primer (Beta-globin) 1
DNA template 3
Nuclease Free Water 25
Table (3): Thermocyclar program for detection of S.pneumoniae
Steps Temperature (°C ) Time Cycle
Initial Denaturation 94 5mints 1
Denaturation 94 30 seconds
Annealing 58 30 seconds 35
Extension 72 30 seconds
Final extension 72 7 mints 1
Detection of S.pneumoniaevirulence factor
- Primers used for ofS.pneumoniaevirulence factors: as in table (4) Table (4): Primers used for ofS.pneumoniaevirulence genes
Gene name Sequences Out product
References PlyA F 5'-ATTTCTGTAACAGCTACCAACGA-3' 329 Saloet
LytA F 5’-CCATTATCAACAGGTCCTACC-3 187 Abdoli et al.,2020
PsaA F 5’-GCCCTAATAAATTGGAGGATCTAATGA-3’ 114 Abdoli et al.,2020
- PCR Reaction mixture and thermocyclar program used for detection of S.pneumoniaeas in table (5) and table (6).
Table (5) PCR Reaction mixture for detection of S.pneumoniaevirulence factors
Master mix components Amount (μM)
Master Mix 20
Forward prime PlyA 1
Reverse primer PlyA 1
Forward prime LytA 1
Reverse primer LytA 1
Forward prime (PsaA) 1
Reverse primer (PsaA) 1
DNA template 3
Nuclease Free Water 21
Table (6): Thermocyclar program for detection of S.pneumoniaevirulence factors
Steps Temperature (°C ) Time Cycle
Initial Denaturation 94 2mints 1
Denaturation 94 15 seconds
Annealing 58 15 seconds 35
Extension 72 50 seconds
Final extension 72 1 mints 1
Results and discussion:
From table (7) and figure (1) showed that Staphylococcus aureusand Streptococcus pneumonia diagnosed in rate of 11.6% and 7.1% respectively . while Klebseilla pneumonia, Pseudomonas aeruginosaand Escherichia coli 6.6%, 6.6% and 3.3% respectively
Table (7): results of bacterial culture and PCR test Spp. of bacteria Number of isolates Isolation ratio
Staphylococcus aureus 9 15%
Streptococcus pneumonia 7 11.6%
Klebseillapneumoniae 4 6.6%
Pseudomonas aeruginosa 4 6.6%
Escherichia coli 2 3.3%
In the current study showed that Streptococcus pneumonia detected in rate of 10%, this results disagreed with results of (Motaweq, et al., 2015) whom recorded isolation rate 12.3% in Nagaf province
And result recorded by (Al Ghizawi et al., 2007) in basrah which are 19% for all streptococcus spp. Hassan&Majid. (2018) detection of that Streptococcus pneumonia in Sulaymaniyah Province in rate of 13% . Saleh, Jarullah,. (2019). Isolated of Streptococcus pneumoniae in rate of 27% Thi-Qar province.
Figure (1): Gel electrophoresis of PCR out product. M: 100bp DNA marker , lanes 1-5 posative sample of S.pneumoniaewith out product in size 268 bp fragment
From table (8) and figure (2) showed that the PlyA&LytAdetected in rate of 100% while PsaAdetected in rate of 71.4%
Table (8): S.pneumoniaevirulence gene detected in current study
Gene No of isolate Ratio
PlyA 7 100%
LytA 7 100%
PsaA 5 71.4%
Figure (2): Result of mPCR for detection of S.pneumoniaevirulence factors , M: 100bp DNA marker , lanes 1,2,3&6 isolates have PlyA gene, LytA gene and PsaAgene with fragment in size
329,187 and 114 respectively. 4&5 : isolates have PlyA gene, LytA gene with fragment in size 329 and 114 respectively
S.pneumoniae intracellular toxin play important roles in pathogenesis. PlyA is a single chain thiol activated protein with 53kDa, consists from 471 amino acid. (Inomata et al.,2020) The main action of PlyA are autolysis of the cell (respiratory system cell as exempla), hemolysin to blood cells and decompose any eukaryotic cell containing the cholesterol (Joshi et al.,2020) LytAgene detection in rate 100%, many studies detection this gene Abdeldaimet al.(2010) and Suzuki et al.(2006) with different in detection rate. S.pneumoniae have three autolysin enzymes which are LytA, LytB, LytC, these enzymes able to distraction of bacterial peptidoglycan and lysis the cells (Ross,2010)
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