Limits...
High Glucose Concentration Promotes Vancomycin-Enhanced Biofilm Formation of Vancomycin-Non-Susceptible Staphylococcus aureus in Diabetic Mice.

Hsu CY, Shu JC, Lin MH, Chong KY, Chen CC, Wen SM, Hsieh YT, Liao WT - PLoS ONE (2015)

Bottom Line: To address this question, the diabetic mouse model infected by vancomycin-resistant S. aureus (VRSA) was used under vancomycin treatment.A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice.By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms in vitro through the increased production of proteases and DNases from VRSA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, No. 259, Wenhua 1st Road, Guishan, Taoyuan 333, Taiwan.

ABSTRACT
We previously demonstrated that vancomycin treatment increased acquisition of eDNA and enhanced biofilm formation of drug-resistant Staphylococcus aureus through a cidA-mediated autolysis mechanism. Recently we found that such enhancement became more significant under a higher glucose concentration in vitro. We propose that besides improper antibiotic treatment, increased glucose concentration environment in diabetic animals may further enhance biofilm formation of drug-resistant S. aureus. To address this question, the diabetic mouse model infected by vancomycin-resistant S. aureus (VRSA) was used under vancomycin treatment. The capacity to form biofilms was evaluated through a catheter-associated biofilm assay. A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice. By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms in vitro through the increased production of proteases and DNases from VRSA. Our study highlights the potentially important role of increased glucose concentration in enhancing biofilm formation in vancomycin-treated diabetic mice infected by drug-resistant S. aureus.

No MeSH data available.


Related in: MedlinePlus

Investigation of the mechanisms underlying vancomycin-triggered biofilm degradation in the absence of glucose.(A) Time courses of PIA production by VRSA strain SJC1200 in different media with/without vancomycin treatment. (B) Evaluation of bacteria-secreted DNase activity by visualizing DNA degradation after incubating a DNA product (861 bp) with the supernatant removed from different VRSA culture conditions. 12 hr and 24 hr: supernatant from a 12-h and a 24-h culture system, respectively. M: a 100-bp DNA ladder marker. P and N: positive and negative controls, respectively. (C) Detection of changes in the transcription levels of cidA and agrA in VRSA by qRT-PCR upon different treatments. The fold change of each transcript was compared with vancomycin-untreated samples at T0. (D) Detection of the bacteria-secreted protease activity by the evaluation of the biofilm degradation (suppression) potential using a static biofilm assay in the absence (Non-PI) or presence (PI) of a protease inhibitor cocktail.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526670&req=5

pone.0134852.g005: Investigation of the mechanisms underlying vancomycin-triggered biofilm degradation in the absence of glucose.(A) Time courses of PIA production by VRSA strain SJC1200 in different media with/without vancomycin treatment. (B) Evaluation of bacteria-secreted DNase activity by visualizing DNA degradation after incubating a DNA product (861 bp) with the supernatant removed from different VRSA culture conditions. 12 hr and 24 hr: supernatant from a 12-h and a 24-h culture system, respectively. M: a 100-bp DNA ladder marker. P and N: positive and negative controls, respectively. (C) Detection of changes in the transcription levels of cidA and agrA in VRSA by qRT-PCR upon different treatments. The fold change of each transcript was compared with vancomycin-untreated samples at T0. (D) Detection of the bacteria-secreted protease activity by the evaluation of the biofilm degradation (suppression) potential using a static biofilm assay in the absence (Non-PI) or presence (PI) of a protease inhibitor cocktail.

Mentions: The expression level of selected major factors involved in matrix accumulation and degradation was measured for characterizing the vancomycin effect. In the absence of vancomycin, the expression of PIA was higher when bacterial cells were cultured in BHIhg than in the other two media. However, the expression of PIA was significantly stronger in the presence of vancomycin, regardless of the presence of glucose (Fig 5A). Therefore, expression of PIA was not the cause of biofilm degradation. The activity of extracellular nuclease was measured to evaluate the potential of a secreted nuclease for degrading biofilm-bound eDNA. A higher nuclease activity was observed in the supernatant removed from BHI, both in 12 h and 24 h cultures compared to the supernatant from BHIg, regardless of the presence of vancomycin. Among samples from 24 h cultures, a 77%, 36%, 16%, and 11% decrease in the DNA load was observed in BHI/V0, BHI/V32, BHIg/V0, and BHIg/V32, respectively (Fig 5B). This result suggests that the release of nuclease by S. aureus is enhanced in the absence of glucose whereas vancomycin may not contribute to the nuclease release. Release of eDNA was evaluated by measuring the transcriptional level of cidA. Consistent with previous findings, vancomycin significantly enhanced cidA expression when bacterial cells were cultured in BHIg, and the effect was even stronger in BHIhg [5]. However, there was no significant difference in cidA expression between vancomycin-treated and-untreated cells cultured in BHI at each of the time points (Fig 5C). This result implies that release of eDNA is suppressed in the absence of glucose regardless the presence of vancomycin. Different types of PSMs which are regulated by the global regulator Agr, have been reported to be involved in biofilm degradation [10]. Thus, expression levels of agrA were measured by qRT-PCR. A slight suppression of the expression level of agrA was detected in BHI and BHIg upon vancomycin treatment. However, a significant increase in agrA expression in BHIhg following vancomycin treatment at each time point was observed in comparison to the vancomycin-free counterparts (Fig 5C). The activity of proteases was evaluated by the addition of a protease inhibitor cocktail to the culture media. The inhibitors significantly reduced vancomycin-enhanced biofilm degradation capacity in BHI suggesting that vnacomycin promoted the expression of protease in the absence of glucose (Fig 5D).


High Glucose Concentration Promotes Vancomycin-Enhanced Biofilm Formation of Vancomycin-Non-Susceptible Staphylococcus aureus in Diabetic Mice.

Hsu CY, Shu JC, Lin MH, Chong KY, Chen CC, Wen SM, Hsieh YT, Liao WT - PLoS ONE (2015)

Investigation of the mechanisms underlying vancomycin-triggered biofilm degradation in the absence of glucose.(A) Time courses of PIA production by VRSA strain SJC1200 in different media with/without vancomycin treatment. (B) Evaluation of bacteria-secreted DNase activity by visualizing DNA degradation after incubating a DNA product (861 bp) with the supernatant removed from different VRSA culture conditions. 12 hr and 24 hr: supernatant from a 12-h and a 24-h culture system, respectively. M: a 100-bp DNA ladder marker. P and N: positive and negative controls, respectively. (C) Detection of changes in the transcription levels of cidA and agrA in VRSA by qRT-PCR upon different treatments. The fold change of each transcript was compared with vancomycin-untreated samples at T0. (D) Detection of the bacteria-secreted protease activity by the evaluation of the biofilm degradation (suppression) potential using a static biofilm assay in the absence (Non-PI) or presence (PI) of a protease inhibitor cocktail.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526670&req=5

pone.0134852.g005: Investigation of the mechanisms underlying vancomycin-triggered biofilm degradation in the absence of glucose.(A) Time courses of PIA production by VRSA strain SJC1200 in different media with/without vancomycin treatment. (B) Evaluation of bacteria-secreted DNase activity by visualizing DNA degradation after incubating a DNA product (861 bp) with the supernatant removed from different VRSA culture conditions. 12 hr and 24 hr: supernatant from a 12-h and a 24-h culture system, respectively. M: a 100-bp DNA ladder marker. P and N: positive and negative controls, respectively. (C) Detection of changes in the transcription levels of cidA and agrA in VRSA by qRT-PCR upon different treatments. The fold change of each transcript was compared with vancomycin-untreated samples at T0. (D) Detection of the bacteria-secreted protease activity by the evaluation of the biofilm degradation (suppression) potential using a static biofilm assay in the absence (Non-PI) or presence (PI) of a protease inhibitor cocktail.
Mentions: The expression level of selected major factors involved in matrix accumulation and degradation was measured for characterizing the vancomycin effect. In the absence of vancomycin, the expression of PIA was higher when bacterial cells were cultured in BHIhg than in the other two media. However, the expression of PIA was significantly stronger in the presence of vancomycin, regardless of the presence of glucose (Fig 5A). Therefore, expression of PIA was not the cause of biofilm degradation. The activity of extracellular nuclease was measured to evaluate the potential of a secreted nuclease for degrading biofilm-bound eDNA. A higher nuclease activity was observed in the supernatant removed from BHI, both in 12 h and 24 h cultures compared to the supernatant from BHIg, regardless of the presence of vancomycin. Among samples from 24 h cultures, a 77%, 36%, 16%, and 11% decrease in the DNA load was observed in BHI/V0, BHI/V32, BHIg/V0, and BHIg/V32, respectively (Fig 5B). This result suggests that the release of nuclease by S. aureus is enhanced in the absence of glucose whereas vancomycin may not contribute to the nuclease release. Release of eDNA was evaluated by measuring the transcriptional level of cidA. Consistent with previous findings, vancomycin significantly enhanced cidA expression when bacterial cells were cultured in BHIg, and the effect was even stronger in BHIhg [5]. However, there was no significant difference in cidA expression between vancomycin-treated and-untreated cells cultured in BHI at each of the time points (Fig 5C). This result implies that release of eDNA is suppressed in the absence of glucose regardless the presence of vancomycin. Different types of PSMs which are regulated by the global regulator Agr, have been reported to be involved in biofilm degradation [10]. Thus, expression levels of agrA were measured by qRT-PCR. A slight suppression of the expression level of agrA was detected in BHI and BHIg upon vancomycin treatment. However, a significant increase in agrA expression in BHIhg following vancomycin treatment at each time point was observed in comparison to the vancomycin-free counterparts (Fig 5C). The activity of proteases was evaluated by the addition of a protease inhibitor cocktail to the culture media. The inhibitors significantly reduced vancomycin-enhanced biofilm degradation capacity in BHI suggesting that vnacomycin promoted the expression of protease in the absence of glucose (Fig 5D).

Bottom Line: To address this question, the diabetic mouse model infected by vancomycin-resistant S. aureus (VRSA) was used under vancomycin treatment.A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice.By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms in vitro through the increased production of proteases and DNases from VRSA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, No. 259, Wenhua 1st Road, Guishan, Taoyuan 333, Taiwan.

ABSTRACT
We previously demonstrated that vancomycin treatment increased acquisition of eDNA and enhanced biofilm formation of drug-resistant Staphylococcus aureus through a cidA-mediated autolysis mechanism. Recently we found that such enhancement became more significant under a higher glucose concentration in vitro. We propose that besides improper antibiotic treatment, increased glucose concentration environment in diabetic animals may further enhance biofilm formation of drug-resistant S. aureus. To address this question, the diabetic mouse model infected by vancomycin-resistant S. aureus (VRSA) was used under vancomycin treatment. The capacity to form biofilms was evaluated through a catheter-associated biofilm assay. A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice. By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms in vitro through the increased production of proteases and DNases from VRSA. Our study highlights the potentially important role of increased glucose concentration in enhancing biofilm formation in vancomycin-treated diabetic mice infected by drug-resistant S. aureus.

No MeSH data available.


Related in: MedlinePlus