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Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions.

Turonova H, Briandet R, Rodrigues R, Hernould M, Hayek N, Stintzi A, Pazlarova J, Tresse O - Front Microbiol (2015)

Bottom Line: Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process.Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts.These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

View Article: PubMed Central - PubMed

Affiliation: SECALIM UMR1014, Institut National de la Recherche Agronomique Nantes, France ; LUNAM Université, Oniris, Université de Nantes Nantes, France ; Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Czech Republic.

ABSTRACT
During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

No MeSH data available.


Related in: MedlinePlus

The CosR is responsible for biofilm maturation inC. jejuni. Biofilm structure of C. jejuni NCTC 11168 and the cosR overexpressing transformant (TrfcosR) after incubation for 24 and 48 h in MAC (black bars) or OEC (white bars). (A) The CLSM images representing an aerial view of biofilm structures with the shadow projection on the right. (B) TrfcosR biofilm development in comparison to the parental strain expressed as a fold changes of maximum height and biomass volume. Statistical data are presented in Supplementary Tables 4, 5.
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Figure 5: The CosR is responsible for biofilm maturation inC. jejuni. Biofilm structure of C. jejuni NCTC 11168 and the cosR overexpressing transformant (TrfcosR) after incubation for 24 and 48 h in MAC (black bars) or OEC (white bars). (A) The CLSM images representing an aerial view of biofilm structures with the shadow projection on the right. (B) TrfcosR biofilm development in comparison to the parental strain expressed as a fold changes of maximum height and biomass volume. Statistical data are presented in Supplementary Tables 4, 5.

Mentions: A second copy of C. jejuni gene cosR and its promoter were inserted into the poorer biofilm-forming strain NCTC 11168 to determine its role in C. jejuni biofilm formation. This construction, with an ectopic copy of the cosR gene and its promoter, enabled to double the expression of the transcript level of cosR in the cells (Supplementary Figure 1) in a same manner as in the cosR-overexpressing strain obtained by Hwang et al. (2011) and used by Oh and Jeon (2014). Then, the parental NCTC 11168 strain and the cosR overexpressing strain, namely transformant (TrfcosR), were compared for their ability to adhere to an inert surface and to develop a biofilm (Figure 4). Using the BioFilm Control Ring Test®, a significantly higher ΔBFI was obtained (P = 0.0007) for the transformed strain, indicating its greater ability to adhere to inert surfaces (Figure 4A). In addition, using the crystal violet assay, the transformant showed enhanced biofilm formation after 24 and 48 h (P = 0.0006 and 0.02, respectively) but not after 72 h (P > 0.05) when compared with its parental strain (Figure 4B). The CLSM observations and biofilm analyses indicated that the transformant formed significantly more (P < 0.01) biofilm than its parental strain (Figure 5, Supplementary Table 4). In addition, the maximum height and biomass volume reached by the transformant was not significantly different from those obtained with the strongest biofilm-forming strain 81-176 (Supplementary Table 5). These data showed that the presence of two genes encoding cosR significantly enhanced biofilm development in MAC (592.7-times higher biomass volume after 24 h). Interestingly, this was correlated with the formation of an open biofilm ultrastructure with voids and water channels similar to the one described for 81-176 (Figures 1, 5A). Comparison of genomic sequences using xBASE2 (Chaudhuri et al., 2008) showed that the cosR gene (cj0335c and cjj0379c, respectively) and its flanking regions are 100% identical in NCTC 11168 and 81-176. Both strains carry the exact same form of the gene. Therefore, some other mechanisms, related to the cosR sequence and its flanking regions, for regulating the C. jejuni biofilm formation, should exist. Moreover, unlike the two wild strains, an increased O2 concentration during cultivation did not promote biofilm formation of the transformant (Figure 5). These data indicate that a second ectopic copy of cosR enhanced biofilm development by promoting a complex architecture of C. jejuni biofilm irrespective of O2 demand. Nevertheless, further experiments should be performed to evaluate cosR transcript level and CosR expression throughout all phases of biofilm development.


Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions.

Turonova H, Briandet R, Rodrigues R, Hernould M, Hayek N, Stintzi A, Pazlarova J, Tresse O - Front Microbiol (2015)

The CosR is responsible for biofilm maturation inC. jejuni. Biofilm structure of C. jejuni NCTC 11168 and the cosR overexpressing transformant (TrfcosR) after incubation for 24 and 48 h in MAC (black bars) or OEC (white bars). (A) The CLSM images representing an aerial view of biofilm structures with the shadow projection on the right. (B) TrfcosR biofilm development in comparison to the parental strain expressed as a fold changes of maximum height and biomass volume. Statistical data are presented in Supplementary Tables 4, 5.
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Figure 5: The CosR is responsible for biofilm maturation inC. jejuni. Biofilm structure of C. jejuni NCTC 11168 and the cosR overexpressing transformant (TrfcosR) after incubation for 24 and 48 h in MAC (black bars) or OEC (white bars). (A) The CLSM images representing an aerial view of biofilm structures with the shadow projection on the right. (B) TrfcosR biofilm development in comparison to the parental strain expressed as a fold changes of maximum height and biomass volume. Statistical data are presented in Supplementary Tables 4, 5.
Mentions: A second copy of C. jejuni gene cosR and its promoter were inserted into the poorer biofilm-forming strain NCTC 11168 to determine its role in C. jejuni biofilm formation. This construction, with an ectopic copy of the cosR gene and its promoter, enabled to double the expression of the transcript level of cosR in the cells (Supplementary Figure 1) in a same manner as in the cosR-overexpressing strain obtained by Hwang et al. (2011) and used by Oh and Jeon (2014). Then, the parental NCTC 11168 strain and the cosR overexpressing strain, namely transformant (TrfcosR), were compared for their ability to adhere to an inert surface and to develop a biofilm (Figure 4). Using the BioFilm Control Ring Test®, a significantly higher ΔBFI was obtained (P = 0.0007) for the transformed strain, indicating its greater ability to adhere to inert surfaces (Figure 4A). In addition, using the crystal violet assay, the transformant showed enhanced biofilm formation after 24 and 48 h (P = 0.0006 and 0.02, respectively) but not after 72 h (P > 0.05) when compared with its parental strain (Figure 4B). The CLSM observations and biofilm analyses indicated that the transformant formed significantly more (P < 0.01) biofilm than its parental strain (Figure 5, Supplementary Table 4). In addition, the maximum height and biomass volume reached by the transformant was not significantly different from those obtained with the strongest biofilm-forming strain 81-176 (Supplementary Table 5). These data showed that the presence of two genes encoding cosR significantly enhanced biofilm development in MAC (592.7-times higher biomass volume after 24 h). Interestingly, this was correlated with the formation of an open biofilm ultrastructure with voids and water channels similar to the one described for 81-176 (Figures 1, 5A). Comparison of genomic sequences using xBASE2 (Chaudhuri et al., 2008) showed that the cosR gene (cj0335c and cjj0379c, respectively) and its flanking regions are 100% identical in NCTC 11168 and 81-176. Both strains carry the exact same form of the gene. Therefore, some other mechanisms, related to the cosR sequence and its flanking regions, for regulating the C. jejuni biofilm formation, should exist. Moreover, unlike the two wild strains, an increased O2 concentration during cultivation did not promote biofilm formation of the transformant (Figure 5). These data indicate that a second ectopic copy of cosR enhanced biofilm development by promoting a complex architecture of C. jejuni biofilm irrespective of O2 demand. Nevertheless, further experiments should be performed to evaluate cosR transcript level and CosR expression throughout all phases of biofilm development.

Bottom Line: Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process.Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts.These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

View Article: PubMed Central - PubMed

Affiliation: SECALIM UMR1014, Institut National de la Recherche Agronomique Nantes, France ; LUNAM Université, Oniris, Université de Nantes Nantes, France ; Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Czech Republic.

ABSTRACT
During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

No MeSH data available.


Related in: MedlinePlus