Molecular profile of Streptococcus pneumoniae from Nasopharyngeal swabs of patients with pneumonia using RAPD and PCR detection of lytA, PsaA2, and HtrA virulence genes
Keywords:
Streptococcus pneumoniae; Nasopharyngeal swabs; of patients with pneumonia; RAPD technique; PCR detection of lytA, PsaA2, and HtrA.Abstract
The present work does underline the molecular profiling of ten Streptococcus pneumoniae clinical strains isolated from nasopharyngeal swabs of patients suffering from pneumonia by the aid of RAPD technique and molecular detection of three virulence genes namely lytA, PsaA2, and HtrA. Nasopharyngeal swabs taken from patients with pneumonia, consulted a pulmonologist at Nasiriya Hospital in Iraq, were streaked on blood agar plates. Ten clinical strains showed typical Streptococcus pneumoniae colonial morphology: Gram-positive diplococcic, small, grey, moist, and mucoid with typical green zone of α-hemolysis on blood agar plates. All tested clinical strains were sensitive to optochin on Müller Hinton agar plates. Confirmatory tests with VITEK2 system, bile solubility, and catalase test did verify the affiliation of the ten tested clinical strains as Streptococcus pneumoniae. The frequency of occurrence of lytA, PsaA2, and HtrA genes was 100% (n= 10 /10), 100% (n= 10/10), and 100% (n= 10 /10), respectively among the ten S. pneumoniae clinical strains. The RAPD-640 PCR did succeed to categorize the ten S. pneumoniae clinical strains under study into 9 groups strains (9 clades) according to the resultant DNA banding profile for each strain. The present data would greatly suggest that the S. pneumoniae is the most prevailing pathogen among patients with pneumonia within the taken sample size.
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References
Annual epidemiological report 2016. European Centre for
Disease Prevention and Control. 2016.Rose AM, Hall CS, Martinez-Alier N. Aetiology and
management of malnutrition in HIV-positive children.
Arch Dis Child. 2014; 99(6):546- 551.
Munthali T, Chabala C, Chama E, et al. Tuberculosis
caseload in children with severe acute malnutrition
related with high hospital-based mortality in Lusaka,
Zambia. BMC Res Notes. 2017;10(1):206.
van der Poll T, Opal SM. Pathogenesis, treatment, and prevention
of pneumococcal pneumonia.Lancet.2009; 374 (9700):1543-
doi:10.1016/S0140-6736(09)61114-4.
Siegel, S. J. & Weiser, J. N. Mechanisms of bacterial colonization of
the respiratory tract. Annu Rev Microbiol.69,425-444 (2015).
Kadioglu, A., Weiser, J. N., Paton, J. C. & Andrew, P. W. The
role of Streptococcus pneumoniae virulence factors in
host respiratory colonization and disease. Nat Rev
Microbiol. 6, 288-301 (2008).
Sullivan, A., Hunt, E., MacSharry, J. & Murphy, D. M. The
microbiome and the pathophysiology of asthma. Respir
Res. 17, 163 (2016).
Venkataraman, A. et al. Application of a neutral community
model to assess structuring of the human lung
microbiome. MBio. 6, e02284-14 (2015).
Dobay O. Molecular characterisation, antibiotic sensitivity and
serotyping of hungarian Streptococcus pneumoniae
isolates. Acta Microbiol Immunol Hung. 2008.55(4):395-407
Whatmore AM, Dowson CG. The autolysin-encoding gene
(lytA) of Streptococcus pneumoniae displays restricted
allelic variation despite localized recombination events
with genes of pneumococcal bacteriophage encoding cell
wall lytic enzymes. Infect Immun. 1999. 67(9):4551-6.
Courtney HS. Degradation of connective tissue proteins by
serine proteases from Streptococcus pneumoniae.
Biochem Biophys Res Commun .1991.175: 1023-1028.
Dawid S, Sebert ME, Weiser JN .Bacteriocin activity of
Streptococcus pneumoniae is controlled by the serine
protease HtrA via posttranscriptional regulation. J
Bacteriol .2009.191: 1509-1518.
- Hoy B,Geppert T, Boehm M, Reisen F,Plattner P, etal.Distinct
roles of secreted HtrA proteases from gram-negative
pathogens in cleaving the junctional protein and tumor
suppressor E-cadherin. J Biol Chern 287:10115-10120.2012.
Ibrahim YM, Kerr AR, McCluskey J, Mitchell TJ .Role of HtrA
in the virulence and competence of Streptococcus
pneumoniae. Infect Immun. .2004. 72: 3584-3591.
Sebert ME, Palmer LM, Rosenberg M, Weiser JN .Microarray¬
basedidentificationof htrA,a Streptococcuspneumoniae gene
that is regulated by the CiaRH two-component system and
contributes to nasopharyngeal colonization. Infect Immun.
.2002.70: 4059-4067.
Kochan TJ, Dawid S. The HtrA protease of Streptococcus
pneumoniae controls density dependent stimulation of
the bacteriocin blp locus via disruption of pheromonesecretion. J Bacteriol. (2013).
Dintilhac A, Alloing G, Granadel C, Claverys JP. Competence
and virulence of Streptococcus pneumoniae: Adc and
PsaA mutants exhibit a requirement for Zn and Mn
resulting from inactivation of putative ABC metal
permeases. Mol Microbiol. 1997. 25(4):727-39
Novak R, Braun JS, Charpentier E, Tuomanen E. Penicillin
tolerance genes of Streptococcus pneumoniae: the ABCtype manganese permease complex Psa. Mol Microbiol.
; 29(5):1285-96.
Berry AM, Paton JC. Sequence heterogeneity of PsaA, a 37-
kilodalton putative adhesin essential for virulence of
Streptococcus pneumoniae. Infect Immun. 1996.
(12):5255-62.
Marra A, Lawson S, Asundi JS, Brigham D, Hromockyj AE. In
vivo characterization of the psa genes from
Streptococcus pneumoniae in multiple models of
infection. Microbiology. 2002.148(Pt 5):1483-91.
Jenkinson, H.F. Cell surface protein receptors in oral
streptococci. FEMS Microbiol Lett 121:133-140.1994.
Suzuki N, Yuyama M, Maeda S, Ogawa H, Mashiko K, Kiyoura
Y. Genotypic identification of
presumptive Streptococcus pneumoniae by PCR using
four genes highly specific for S. pneumoniae. J Med
Microbiol 2006; 55: 709- 714
Amies C.R., Can. J. Public Health, 1967, 58:296
MacFaddin J. F., Media for Isolation-CultivationIdentification-Maintenance of Medical Bacteria,
Vol. I, Williams and Wilkins, Baltimore.
Cheesbrough M. District laboratory Practice in Tropical
Countries. Part 2. Cambridge University Press; 2006.
-157 p.
Centers for Disease Control and Prevention. Laboratory
Methods for the Diagnosis of Meningitis. Chapter 8:
Identification and Characterization of Streptococcus
pneumoniae. US: CDC; 2016. 14 p
CLSI. Performance Standards for Antimicrobial
Susceptibility Testing. 28th ed. CLSI supplement M100.
Wayne, PA: Clinical and Laboratory Standards Institute.
:1-282.
Green MR, Sambrook J. Molecular cloning: A Laboratory
Manual. 1989.Cold Spring Herber Laboratory Press.
Suzuki N, Yuyama M, Maeda S, Ogawa H, Mashiko K, Kiyoura
Y. Genotypic identification of
presumptive Streptococcus pneumoniae by PCR using
four genes highly specific for S. pneumoniae. J Med
Microbiol 2006; 55: 709- 714
Zahao W, Pan F, Wang B, Wang C, Sun Y, Zhang T, Shi Y,
Zhang H. Epidemiology Characteristics of Streptococcus
pneumoniae from Children with Pneumonia in Shanghai:
A Retrospective Study. Front. Cell. Infect. Microbiol., 18;
https://doi.org/10.3389/fcimb.2019.00258.
Paton JC, Berry AM, Lock RA. Molecular analysis of putative
pneumococcal virulence proteins.Microb Drug Resist.
; 3(1):1-10.
Martner, A., Dahlgren, C., Paton, J. C., and Wold, A. E. Infect.
Immun. 76, 4079-4087. 2008.
Eldholm, V., Johnsborg, O., Haugen, K., Ohnstad, H. S., and
Havarstein, L. S. (2009) Microbiology 155, 2223-2234
Martner, A., Skovbjerg, S., Paton, J. C., and Wold, A. E. Infect.
Immun. 2009. 77, 3826-3837.
Zhang GW, Kotiw M, Daggard G. A RAPD-PCR genotyping
assay which correlates with serotypes of group B
streptococci. Lett Appl Microbiol. 2002; 35(3):247-50.
doi: 1O.1O46/j.1472-765x.2OO2.01177. x.
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