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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%.

Introduction

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.

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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

Beta- globin

F 5’-GAAGAGCCAAGGACAGGTAC-3’ 268 Jourdain et

al.,2011

R 5’-CAACTTCATCCACGTTCACC-3’

- 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

Total 50

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

size

References PlyA F 5'-ATTTCTGTAACAGCTACCAACGA-3' 329 Saloet

al.,1995

R 5'-GAATTCCCTGTCTTTTCAAAGTC-3'

LytA F 5’-CCATTATCAACAGGTCCTACC-3 187 Abdoli et al.,2020

R 5’-TAAGAACAGATTTGCCTCAAG-3’

PsaA F 5’-GCCCTAATAAATTGGAGGATCTAATGA-3’ 114 Abdoli et al.,2020

R 5’-GACCAGAAGTTGTATCTTTTTTTCCG-3’

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- 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

Total 50

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.

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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

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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)

References

1- Abdeldaim, G.;Herrmann, B.; Mölling, P.; Holmberg, H.; Blomberg, J.; Olcén, P. and Strålin, K. (2010). Usefulness of real‐time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia. Clinical Microbiology and Infection, 16(8):1135-1141.

2- Abdoli, S., Safamanesh, S., Khosrojerdi, M., &Azimian, A. (2020). Molecular detection and serotyping of Streptococcus pneumoniae in children with suspected meningitis in Northeast Iran. Iranian journal of medical sciences, 45(2), 125.‏

3- Al-Ghizawi, G. J.; Al-Sulami, A. A. and Al-Taher, S. S. (2007). Profile of community-and hospital-acquired pneumonia cases admitted to Basrah general hospital, Iraq. Eastern Mediterranean Health Journal, 13(2): 230-242.

4- Aykac, K., Ozsurekci, Y., CuraYayla, B. C., Evren, K., LacinelGurlevik, S., Oygar, P. D., ...

& Ceyhan, M. (2021). Pneumococcal carriage in children with COVID-19. Human Vaccines

&Immunotherapeutics, 17(6), 1628-1634.‏

5- D'Mello, A. (2021). Identification of core genes involved in Streptococcus pneumoniae host- pathogen interactions under diverse infections, and their potential as therapeutic targets (Doctoral dissertation).‏

6- Hassan, T. M., &Majid, B. T. (2018). Identification of Common Aerobic Bacterial Isolates among Conjunctivitis in Sulaymaniyah Province/Iraq. Iraqi Journal of Medical Sciences, 16(2).‏

7- Inomata, M., Xu, S., Chandra, P., Meydani, S. N., Takemura, G., Philips, J. A., & Leong, J.

M. (2020). Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging. Proceedings of the National Academy of Sciences, 117(52), 33561-33569.‏

8- Joshi, B., Singh, B., Nadeem, A., Askarian, F., Wai, S. N., Johannessen, M., &Hegstad, K.

(2020). Transcriptome profiling of Staphylococcusaureus associated extracellular vesicles reveals presence of small RNA-cargo. Frontiers in molecular biosciences, 7, 482.‏

9- Jourdain S, Dreze PA, Vandeven J, Verhaegen J, Van Melderen L, Smeesters PR.

Sequential multiplex PCR assay for determining capsular serotypes of colonizing S.

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pneumoniae. BMC Infect Dis. 2011;11:100. doi: 10.1186/1471-2334-11-100. PubMed PMID: 21507244; PubMed Central PMCID: PMCPMC3094224.

10- Motaweq, Z.Y.; Naher, H.S. and Al-Dahhan, H.A. (2015). Phenotypic and genotypic characterization of some virulence factors in Streptococcus pneumoniaeisolated from patients with LRTI in Najaf Province/ Iraq. International Journal of Scientific and Engineering Research, 6(8): 459-465.

11- Saleh, M. B., &Jarullah, B. A. (2019). Isolation and Identification of Streptococcus pneumoniae isolated from Pneumonia patients in Thi-Qar province/Iraq. Journal of Education for Pure Science, 9(1).‏

12- Salo, P.; Örtqvist, Å. and Leinonen, M. (1995). Diagnosis of bacteremic pneumococcal pneumonia by amplification of pneumolysin gene fragment in serum. Journal of Infectious Diseases, 171(2): 479-482.

13- Schulze, A., Mitterer, F., Pombo, J. P., &Schild, S. (2021). Biofilms by bacterial human pathogens: Clinical relevance-development, composition and regulation-therapeutical strategies. Microbial Cell, 8(2), 28.‏

14- Suzuki, N.; Yuyama, M.;Maeda, S.; Ogawa, H.; Mashiko, K. and Kiyoura, Y. (2006).

Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniae. Journal of Medical Microbiology, 55(6):709-714.

15- Weisbroth, S. H., & Kohn, D. F. (2020). Bacterial, mycoplasmal, and mycotic infections. The laboratory rat, 451-539.‏

16- Welp, A. L., &Bomberger, J. M. (2020). Bacterial community interactions during chronic respiratory disease. Frontiers in Cellular and Infection Microbiology, 10, 213.‏

17- Ross, K. S. (2010). Novel strategies to prevent and treat experimental pneumococcal disease (Doctoral dissertation, University of Glasgow).‏

Referințe

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