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Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Repizo GD, Gagné S, Foucault-Grunenwald ML, Borges V, Charpentier X, Limansky AS, Gomes JP, Viale AM, Salcedo SP - PLoS ONE (2015)

Bottom Line: The T6SS genomic locus is present and was actively transcribed in all of the above strains.In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections.Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, CNRS UMR 5086, Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France.

ABSTRACT
Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

No MeSH data available.


Related in: MedlinePlus

A. baumannii strain-specific growth characteristics and biofilm formation.A) The different strains were grown over-night in TSB rich medium (upper panel) or BM2 minimal medium supplemented with 10 mM glutamate (BM2G; middle panel). Bottom panel corresponds to polystyrene wells microtiter plates labelled with crystal violet and quantification is shown in B). The results are expressed as means ± SEM of experiments performed in triplicate (**** P<0.0001). The difference between the DSM30011 and each of the clinical isolates was also significant (**** P<0.0001) but not shown in the graph for simplicity. C) Confocal microscope 3D reconstruction of biofilm formed by the different strains, formed on glass-bottom slides after 24 h in static conditions. Images correspond to a slice obtained from each biofilm stack, labelled with DAPI. Depth analysis is shown for the 3D reconstruction of the z-stacks obtained for each strain. D) Analysis of the biofilm forming capacities of wild-type DSM30011, ΔtssM, and ΔtssMptssM-complemented strains grown for 24 h in BM2G medium following crystal violet staining.
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pone.0138265.g006: A. baumannii strain-specific growth characteristics and biofilm formation.A) The different strains were grown over-night in TSB rich medium (upper panel) or BM2 minimal medium supplemented with 10 mM glutamate (BM2G; middle panel). Bottom panel corresponds to polystyrene wells microtiter plates labelled with crystal violet and quantification is shown in B). The results are expressed as means ± SEM of experiments performed in triplicate (**** P<0.0001). The difference between the DSM30011 and each of the clinical isolates was also significant (**** P<0.0001) but not shown in the graph for simplicity. C) Confocal microscope 3D reconstruction of biofilm formed by the different strains, formed on glass-bottom slides after 24 h in static conditions. Images correspond to a slice obtained from each biofilm stack, labelled with DAPI. Depth analysis is shown for the 3D reconstruction of the z-stacks obtained for each strain. D) Analysis of the biofilm forming capacities of wild-type DSM30011, ΔtssM, and ΔtssMptssM-complemented strains grown for 24 h in BM2G medium following crystal violet staining.

Mentions: When routinely cultivating the analyzed A. baumannii strains in liquid culture media we repeatedly observed that DSM30011 cells have a higher tendency to grow on the liquid-air interface forming a thick pellicle on the surface. This observation was confirmed by analyzing their growth characteristics in both rich (TSB) and glutamate supplemented minimal (BM2G) liquid media (Fig 6A). We then investigated the ability of the different A. baumannii strains to produce biofilm, thought to provide a reservoir for transmission of A. baumannii to susceptible patients in hospital surfaces [4]. Following 24 h incubation, the DSM30011 strain exhibited the highest ability to adhere to polystyrene surfaces suggesting biofilm production (Fig 6A and 6B). In agreement with previous work [29], we observed that ATCC17978 is not a strong biofilm producer, a situation also observed for the clinical strains analysed which produced similar or even lower amounts of biofilm than this reference strain (Fig 6A and 6B). Confocal microscopy confirmed the ability of A. baumannii DSM30011 cells to form a biofilm with numerous large bacterial aggregates visible after 24 h (S4 Fig). On the contrary, this was not found for the ATCC17978 strain (S4 Fig) or the clinical isolates (data not shown). Analysis of the depth of bacterial growth (excluding the bacterial aggregates) on the glass-bottom plates under static growth conditions confirmed extensive biofilm formation in the case of DSM30011 in contrast to ATCC17978 cells (Fig 6C). As the T6SS has been implicated in biofilm formation in other bacterial pathogens [30–32] we investigated the ability of the A. baumannii DSM30011 ΔtssM mutant to adhere to abiotic surfaces and form biofilm. Under static growth conditions no significant differences were observed in comparison to the wild-type or complemented strains by neither microscopy analysis of biofilm depth (Fig 6C) nor following crystal violet staining (Fig 6D), suggesting that the T6SS is not required for biofilm formation in this A. baumannii strain.


Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Repizo GD, Gagné S, Foucault-Grunenwald ML, Borges V, Charpentier X, Limansky AS, Gomes JP, Viale AM, Salcedo SP - PLoS ONE (2015)

A. baumannii strain-specific growth characteristics and biofilm formation.A) The different strains were grown over-night in TSB rich medium (upper panel) or BM2 minimal medium supplemented with 10 mM glutamate (BM2G; middle panel). Bottom panel corresponds to polystyrene wells microtiter plates labelled with crystal violet and quantification is shown in B). The results are expressed as means ± SEM of experiments performed in triplicate (**** P<0.0001). The difference between the DSM30011 and each of the clinical isolates was also significant (**** P<0.0001) but not shown in the graph for simplicity. C) Confocal microscope 3D reconstruction of biofilm formed by the different strains, formed on glass-bottom slides after 24 h in static conditions. Images correspond to a slice obtained from each biofilm stack, labelled with DAPI. Depth analysis is shown for the 3D reconstruction of the z-stacks obtained for each strain. D) Analysis of the biofilm forming capacities of wild-type DSM30011, ΔtssM, and ΔtssMptssM-complemented strains grown for 24 h in BM2G medium following crystal violet staining.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581634&req=5

pone.0138265.g006: A. baumannii strain-specific growth characteristics and biofilm formation.A) The different strains were grown over-night in TSB rich medium (upper panel) or BM2 minimal medium supplemented with 10 mM glutamate (BM2G; middle panel). Bottom panel corresponds to polystyrene wells microtiter plates labelled with crystal violet and quantification is shown in B). The results are expressed as means ± SEM of experiments performed in triplicate (**** P<0.0001). The difference between the DSM30011 and each of the clinical isolates was also significant (**** P<0.0001) but not shown in the graph for simplicity. C) Confocal microscope 3D reconstruction of biofilm formed by the different strains, formed on glass-bottom slides after 24 h in static conditions. Images correspond to a slice obtained from each biofilm stack, labelled with DAPI. Depth analysis is shown for the 3D reconstruction of the z-stacks obtained for each strain. D) Analysis of the biofilm forming capacities of wild-type DSM30011, ΔtssM, and ΔtssMptssM-complemented strains grown for 24 h in BM2G medium following crystal violet staining.
Mentions: When routinely cultivating the analyzed A. baumannii strains in liquid culture media we repeatedly observed that DSM30011 cells have a higher tendency to grow on the liquid-air interface forming a thick pellicle on the surface. This observation was confirmed by analyzing their growth characteristics in both rich (TSB) and glutamate supplemented minimal (BM2G) liquid media (Fig 6A). We then investigated the ability of the different A. baumannii strains to produce biofilm, thought to provide a reservoir for transmission of A. baumannii to susceptible patients in hospital surfaces [4]. Following 24 h incubation, the DSM30011 strain exhibited the highest ability to adhere to polystyrene surfaces suggesting biofilm production (Fig 6A and 6B). In agreement with previous work [29], we observed that ATCC17978 is not a strong biofilm producer, a situation also observed for the clinical strains analysed which produced similar or even lower amounts of biofilm than this reference strain (Fig 6A and 6B). Confocal microscopy confirmed the ability of A. baumannii DSM30011 cells to form a biofilm with numerous large bacterial aggregates visible after 24 h (S4 Fig). On the contrary, this was not found for the ATCC17978 strain (S4 Fig) or the clinical isolates (data not shown). Analysis of the depth of bacterial growth (excluding the bacterial aggregates) on the glass-bottom plates under static growth conditions confirmed extensive biofilm formation in the case of DSM30011 in contrast to ATCC17978 cells (Fig 6C). As the T6SS has been implicated in biofilm formation in other bacterial pathogens [30–32] we investigated the ability of the A. baumannii DSM30011 ΔtssM mutant to adhere to abiotic surfaces and form biofilm. Under static growth conditions no significant differences were observed in comparison to the wild-type or complemented strains by neither microscopy analysis of biofilm depth (Fig 6C) nor following crystal violet staining (Fig 6D), suggesting that the T6SS is not required for biofilm formation in this A. baumannii strain.

Bottom Line: The T6SS genomic locus is present and was actively transcribed in all of the above strains.In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections.Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, CNRS UMR 5086, Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France.

ABSTRACT
Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

No MeSH data available.


Related in: MedlinePlus