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Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Sahu PK, Iyer PS, Barage SH, Sonawane KD, Chopade BA - ScientificWorldJournal (2014)

Bottom Line: Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05).Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h).Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India ; Ispat General Hospital, SAIL, Rourkela 769 005, India.

ABSTRACT
Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

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(a)–(h) Visualization of biofilm augmentation by scanning electron microscopy. Electron micrographs (a, c, e, g) showing biofilms formed by algC clones after 24 h, at a gradient of magnifications (Bar = 2 μm, 5 μm, 10 μm, and 20 μm; absolute magnifications indicated as Kx in the very picture) as observed under a scanning electron microscope. algC clones can be seen clearly producing dense and robust biofilm as suggested by the thickness and integrity of the biofilm matrices, compared to the control biofilms by E. coli DH5α (lacking A. baumannii algC gene) at corresponding comparable magnifications (b, d, f, h).
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fig6: (a)–(h) Visualization of biofilm augmentation by scanning electron microscopy. Electron micrographs (a, c, e, g) showing biofilms formed by algC clones after 24 h, at a gradient of magnifications (Bar = 2 μm, 5 μm, 10 μm, and 20 μm; absolute magnifications indicated as Kx in the very picture) as observed under a scanning electron microscope. algC clones can be seen clearly producing dense and robust biofilm as suggested by the thickness and integrity of the biofilm matrices, compared to the control biofilms by E. coli DH5α (lacking A. baumannii algC gene) at corresponding comparable magnifications (b, d, f, h).

Mentions: Electron micrographs of biofilm formed by the algC clones (Figure 6) showed significant increase in biofilm formation compared to controls, probably indicative of the overexpression of the PMM/PGM protein and the resultant exopolysaccharides. Thicker biofilms could be seen in the algC clones having clear dense matrices (Figures 6(a), 6(c), and 6(e)) with a intracellular cementing material clearly visible (Figures 6(a) and 6(e)) indicative of the biofilm EPS. Overall SEM analysis at a gradient of magnifications and concurrent comparisons with control clones (lacking functional copy of algC) suggested substantially that there is significant facilitation of the overall biofilm formation, emphasizing the enrichment of the biofilm matrices, which could be due to the cohesive activity of EPS including alginate and LPS cores. The quantitative biofilm assay concurred with the microscopic analysis, with augmentation of biofilm up to 3.87-fold in the algC clones compared to the control cells lacking algC gene (P < 0.02, data not shown).


Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Sahu PK, Iyer PS, Barage SH, Sonawane KD, Chopade BA - ScientificWorldJournal (2014)

(a)–(h) Visualization of biofilm augmentation by scanning electron microscopy. Electron micrographs (a, c, e, g) showing biofilms formed by algC clones after 24 h, at a gradient of magnifications (Bar = 2 μm, 5 μm, 10 μm, and 20 μm; absolute magnifications indicated as Kx in the very picture) as observed under a scanning electron microscope. algC clones can be seen clearly producing dense and robust biofilm as suggested by the thickness and integrity of the biofilm matrices, compared to the control biofilms by E. coli DH5α (lacking A. baumannii algC gene) at corresponding comparable magnifications (b, d, f, h).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4269089&req=5

fig6: (a)–(h) Visualization of biofilm augmentation by scanning electron microscopy. Electron micrographs (a, c, e, g) showing biofilms formed by algC clones after 24 h, at a gradient of magnifications (Bar = 2 μm, 5 μm, 10 μm, and 20 μm; absolute magnifications indicated as Kx in the very picture) as observed under a scanning electron microscope. algC clones can be seen clearly producing dense and robust biofilm as suggested by the thickness and integrity of the biofilm matrices, compared to the control biofilms by E. coli DH5α (lacking A. baumannii algC gene) at corresponding comparable magnifications (b, d, f, h).
Mentions: Electron micrographs of biofilm formed by the algC clones (Figure 6) showed significant increase in biofilm formation compared to controls, probably indicative of the overexpression of the PMM/PGM protein and the resultant exopolysaccharides. Thicker biofilms could be seen in the algC clones having clear dense matrices (Figures 6(a), 6(c), and 6(e)) with a intracellular cementing material clearly visible (Figures 6(a) and 6(e)) indicative of the biofilm EPS. Overall SEM analysis at a gradient of magnifications and concurrent comparisons with control clones (lacking functional copy of algC) suggested substantially that there is significant facilitation of the overall biofilm formation, emphasizing the enrichment of the biofilm matrices, which could be due to the cohesive activity of EPS including alginate and LPS cores. The quantitative biofilm assay concurred with the microscopic analysis, with augmentation of biofilm up to 3.87-fold in the algC clones compared to the control cells lacking algC gene (P < 0.02, data not shown).

Bottom Line: Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05).Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h).Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India ; Ispat General Hospital, SAIL, Rourkela 769 005, India.

ABSTRACT
Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

Show MeSH
Related in: MedlinePlus