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Screening of molecular virulence markers in Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from clinical infections.

Cotar AI, Chifiriuc MC, Dinu S, Bucur M, Iordache C, Banu O, Dracea O, Larion C, Lazar V - Int J Mol Sci (2010)

Bottom Line: Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts.The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens.Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Research in Microbiology and Immunology, Cantacuzino, Spl. Independentei 103, cod 060631, Bucharest 060101, Romania; E-Mails: aniioana@yahoo.com (A.-I.C.); carmeniordache78@yahoo.com (C.I.); olgutza_dracea@yahoo.co.uk (O.D.); larioncristina@yahoo.com (C.L.).

ABSTRACT
Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of new strategies in order to elucidate the different mechanisms used by these bacteria at different stages of the infectious process, providing the scientists with new procedures for preventing, or at least improving, the control of S. aureus and Ps. aeruginosa infections. The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens. We used multiplex and uniplex PCR assays to detect the genes encoding different cell-wall associated and extracellular virulence factors, in order to evaluate potential associations between the presence of putative virulence genes and the outcome of infections caused by these bacteria. Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections.

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Related in: MedlinePlus

Ethidium bromide-stained 1.5% agarose gel showing the amplified products of plcN gene. Lines 1, 9 and 14: DNA ladder 100bp (Promega); 2: Ps. aeruginosa 101; 3: Ps. aeruginosa 1558; 4: Ps. aeruginosa 111; 5: Ps. aeruginosa 1443; 6: Ps. aeruginosa 1093; 7: Ps. aeruginosa 1561; 8: negative control (pure water); 10: Ps. aeruginosa 20; 11: Ps. aeruginosa 1442; 12: Ps. aeruginosa 84; and 13: Ps. Aeruginosa 1562.
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f13-ijms-11-05273: Ethidium bromide-stained 1.5% agarose gel showing the amplified products of plcN gene. Lines 1, 9 and 14: DNA ladder 100bp (Promega); 2: Ps. aeruginosa 101; 3: Ps. aeruginosa 1558; 4: Ps. aeruginosa 111; 5: Ps. aeruginosa 1443; 6: Ps. aeruginosa 1093; 7: Ps. aeruginosa 1561; 8: negative control (pure water); 10: Ps. aeruginosa 20; 11: Ps. aeruginosa 1442; 12: Ps. aeruginosa 84; and 13: Ps. Aeruginosa 1562.

Mentions: Three other soluble proteins involved in Ps. aeruginosa invasion are represented by a rhamnolipid, two phospholipases C, haemolytic phospholipase C (PLC-H), and non-haemolytic phospholipase C (PLC-N). These factors may act synergistically to break down phospholipids (e.g., phosphatidylcholine and sphingomyelin), and contribute to invasion by means of their cytotoxic effects on neutrophils, lymphocytes and other eucaryotic cells. Rhamnolipid, a rhamnose-containing glycolipid biosurfactant, has a detergent-like structure and is considered to solubilize the phospholipids of lung surfactant, making them more accessible to cleavage by phospholipase C. The resulting loss of lung surfactant may be responsible for the atelectasis associated with chronic and acute Ps. aeruginosa lung infection. Rhamnolipid also inhibits the mucociliary transport and ciliary function of human respiratory epithelium [13]. The results of PCR analysis showed that all Ps. aeruginosa possess rhlAB gene, encoding the rhamnolipid (Figure 11). Concerning the presence of two phospholipases C, the results of PCR analysis showed that all Ps. aeruginosa strains possess plcN gene, whereas plcH gene is present only in seven of the studied strains (Figures 12 and 13). These two enzymes could work sequentially and synergistically, PLC-H would promote degradation of the erythrocyte membrane (phospholipids components of the outer leaflet: phosphatidylcholine and sphingomyelin), exposing the inner leaflet. PLC-N could then hydrolyze phospatidylserine present in the inner leaflet. The products of phospholipid hydrolysis would be digested by alkaline phosphatase, pyrophosphate being released. The experimental studies demonstrated that purified PLC-H determines vascular permeability, organ injuries, and death when injected in high doses into mice, proving that PLC-H is an important virulence factor. Some Ps. aeruginosa strains are producers of a mucoid exopolysaccharide (alginate) that forms the matrix of the Ps. aeruginosa biofilm, anchoring the cells and, in certain cases, protecting bacteria from the host defense armamentarium consisting of: lymphocytes, phagocytes, the ciliary action of the respiratory tract, antibodies and complement. At the same time these strains are less susceptible to antibiotics than their planktonic counterparts, being most often isolated from patients with cystic fibrosis and usually found in lung tissues of such individuals. The algD gene encodes GDP-mannose dehydrogenase (GMD), which acts as a rate-limiting enzyme in mucoid strains by catalyzing the conversion of GDP-mannose to GDP-mannuronic acid, thereby promoting the cell to alginate production [14]. The results of PCR analysis concerning the presence of algD gene showed that only five strains possess this gene, an aspect which demonstrates the involvement of these strains in infections with biofilm formation (Figure 14).


Screening of molecular virulence markers in Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from clinical infections.

Cotar AI, Chifiriuc MC, Dinu S, Bucur M, Iordache C, Banu O, Dracea O, Larion C, Lazar V - Int J Mol Sci (2010)

Ethidium bromide-stained 1.5% agarose gel showing the amplified products of plcN gene. Lines 1, 9 and 14: DNA ladder 100bp (Promega); 2: Ps. aeruginosa 101; 3: Ps. aeruginosa 1558; 4: Ps. aeruginosa 111; 5: Ps. aeruginosa 1443; 6: Ps. aeruginosa 1093; 7: Ps. aeruginosa 1561; 8: negative control (pure water); 10: Ps. aeruginosa 20; 11: Ps. aeruginosa 1442; 12: Ps. aeruginosa 84; and 13: Ps. Aeruginosa 1562.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100824&req=5

f13-ijms-11-05273: Ethidium bromide-stained 1.5% agarose gel showing the amplified products of plcN gene. Lines 1, 9 and 14: DNA ladder 100bp (Promega); 2: Ps. aeruginosa 101; 3: Ps. aeruginosa 1558; 4: Ps. aeruginosa 111; 5: Ps. aeruginosa 1443; 6: Ps. aeruginosa 1093; 7: Ps. aeruginosa 1561; 8: negative control (pure water); 10: Ps. aeruginosa 20; 11: Ps. aeruginosa 1442; 12: Ps. aeruginosa 84; and 13: Ps. Aeruginosa 1562.
Mentions: Three other soluble proteins involved in Ps. aeruginosa invasion are represented by a rhamnolipid, two phospholipases C, haemolytic phospholipase C (PLC-H), and non-haemolytic phospholipase C (PLC-N). These factors may act synergistically to break down phospholipids (e.g., phosphatidylcholine and sphingomyelin), and contribute to invasion by means of their cytotoxic effects on neutrophils, lymphocytes and other eucaryotic cells. Rhamnolipid, a rhamnose-containing glycolipid biosurfactant, has a detergent-like structure and is considered to solubilize the phospholipids of lung surfactant, making them more accessible to cleavage by phospholipase C. The resulting loss of lung surfactant may be responsible for the atelectasis associated with chronic and acute Ps. aeruginosa lung infection. Rhamnolipid also inhibits the mucociliary transport and ciliary function of human respiratory epithelium [13]. The results of PCR analysis showed that all Ps. aeruginosa possess rhlAB gene, encoding the rhamnolipid (Figure 11). Concerning the presence of two phospholipases C, the results of PCR analysis showed that all Ps. aeruginosa strains possess plcN gene, whereas plcH gene is present only in seven of the studied strains (Figures 12 and 13). These two enzymes could work sequentially and synergistically, PLC-H would promote degradation of the erythrocyte membrane (phospholipids components of the outer leaflet: phosphatidylcholine and sphingomyelin), exposing the inner leaflet. PLC-N could then hydrolyze phospatidylserine present in the inner leaflet. The products of phospholipid hydrolysis would be digested by alkaline phosphatase, pyrophosphate being released. The experimental studies demonstrated that purified PLC-H determines vascular permeability, organ injuries, and death when injected in high doses into mice, proving that PLC-H is an important virulence factor. Some Ps. aeruginosa strains are producers of a mucoid exopolysaccharide (alginate) that forms the matrix of the Ps. aeruginosa biofilm, anchoring the cells and, in certain cases, protecting bacteria from the host defense armamentarium consisting of: lymphocytes, phagocytes, the ciliary action of the respiratory tract, antibodies and complement. At the same time these strains are less susceptible to antibiotics than their planktonic counterparts, being most often isolated from patients with cystic fibrosis and usually found in lung tissues of such individuals. The algD gene encodes GDP-mannose dehydrogenase (GMD), which acts as a rate-limiting enzyme in mucoid strains by catalyzing the conversion of GDP-mannose to GDP-mannuronic acid, thereby promoting the cell to alginate production [14]. The results of PCR analysis concerning the presence of algD gene showed that only five strains possess this gene, an aspect which demonstrates the involvement of these strains in infections with biofilm formation (Figure 14).

Bottom Line: Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts.The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens.Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Research in Microbiology and Immunology, Cantacuzino, Spl. Independentei 103, cod 060631, Bucharest 060101, Romania; E-Mails: aniioana@yahoo.com (A.-I.C.); carmeniordache78@yahoo.com (C.I.); olgutza_dracea@yahoo.co.uk (O.D.); larioncristina@yahoo.com (C.L.).

ABSTRACT
Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of new strategies in order to elucidate the different mechanisms used by these bacteria at different stages of the infectious process, providing the scientists with new procedures for preventing, or at least improving, the control of S. aureus and Ps. aeruginosa infections. The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens. We used multiplex and uniplex PCR assays to detect the genes encoding different cell-wall associated and extracellular virulence factors, in order to evaluate potential associations between the presence of putative virulence genes and the outcome of infections caused by these bacteria. Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections.

Show MeSH
Related in: MedlinePlus