Staphylococcus epidermidis biofilms with higher proportions of dormant bacteria induce a lower activation of murine macrophages - PubMed (original) (raw)

Staphylococcus epidermidis biofilms with higher proportions of dormant bacteria induce a lower activation of murine macrophages

Filipe Cerca et al. J Med Microbiol. 2011 Dec.

Abstract

Staphylococcus epidermidis is an opportunistic pathogen and, due to its ability to establish biofilms, is a leading causative agent of indwelling medical device-associated infection. The presence of high amounts of dormant bacteria is a hallmark of biofilms, making them more tolerant to antimicrobials and to the host immune response. We observed that S. epidermidis biofilms grown in excess glucose accumulated high amounts of viable but non-culturable (VBNC) bacteria, as assessed by their low ratio of culturable bacteria over the number of viable bacteria. This effect, which was a consequence of the accumulation of acidic compounds due to glucose metabolism, was counteracted by high extracellular levels of calcium and magnesium added to the culture medium allowing modulation of the proportions of VBNC bacteria within S. epidermidis biofilms. Using bacterial inocula obtained from biofilms with high and low proportions of VBNC bacteria, their stimulatory effect on murine macrophages was evaluated in vitro and in vivo. The inoculum enriched in VBNC bacteria induced in vitro a lower production of tumour necrosis factor alpha, interleukin-1 and interleukin-6 by bone-marrow-derived murine macrophages and, in vivo, a lower stimulatory effect on peritoneal macrophages, assessed by increased surface expression of Gr1 and major histocompatibility complex class II molecules. Overall, these results show that environmental conditions, such as pH and extracellular levels of calcium and magnesium, can induce dormancy in S. epidermidis biofilms. Moreover, they show that bacterial suspensions enriched in dormant cells are less inflammatory, suggesting that dormancy can contribute to the immune evasion of biofilms.

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Figures

Fig. 1.

Glucose induces cell dormancy in S. epidermidis biofilms. (a) The number of c.f.u. over the number of viable bacteria obtained from biofilms grown as indicated shown as percentages. Results are a representative example of four independent experiments. Statistically significant differences (P<0.01, ANOVA) are indicated by asterisks (**). (b, c) Determination of (b) lactic acid accumulation and (c) culture medium pH over time in biofilms grown in TSB (⧫) or TSB 1 %G (□). Results represent the mean value±

and are a representative example of three independent experiments.

Fig. 2.

Effect of calcium and magnesium on the glucose-induced cell dormancy. (a) The number of c.f.u. over the number of viable bacteria obtained from biofilms grown as indicated shown as percentages. Results are a representative example of four independent experiments. Statistically significant differences between groups (P<0.01, ANOVA) are indicated by asterisks (**). Numbers of viable and culturable bacteria within each biofilm grown as described are also presented. (b, c) Determination of (b) glucose consumption and (c) lactic acid accumulation over time in cultures of biofilms grown in TSB 1 %G (□) or TSB 1 %G+Ca2+/Mg2+ (⧫). Results represent the mean value±

and are a representative example of three independent experiments. (d) The number of c.f.u. over the number of viable bacteria obtained from S. epidermidis PE9, M187, JI6 and IE86 biofilms grown in TSB 1 %G (black bars) or TSB 1 %G+Ca2+/Mg2+(grey bars) shown as percentages. Results are a representative example of two independent experiments. Statistically significant differences between groups (P<0.01, ANOVA) are indicated by asterisks (**).

Fig. 3.

Effect of cell dormancy on BMDM activation. (a) Concentrations of TNF-α, IL-1 and IL-6 in cell culture supernatants of BMDMs after 9 h incubation with 1×104 live bacteria obtained from _S. epidermidis_biofilms grown as indicated. Black bars, TSB; hatched bars, TSB+Ca2+/Mg2+; grey bars, TSB 1%G; white bars, TSB 1%G+Ca2+/Mg2+. (b) Numbers of c.f.u. in BMDM culture supernatants determined at 3 h intervals after bacterial challenge. Results are a representative example of three independent experiments. ⧫, TSB; ○, TSB+Ca2+/Mg2+; ▴, TSB 1%G; ▪, TSB 1%G+Ca2+/Mg2+. The statistically significant difference between the TSB 1 %G inoculum and the other inocula (P<0.01, ANOVA) is indicated by asterisks (**).

Fig. 4.

Evaluation by flow cytometric analysis of BMDM death following 2 h incubation with intact S. epidermidis biofilms grown as indicated. BMDMs were discriminated from bacteria according to the SSC parameter. Dot plots are representative examples of two independent experiments. The mean proportion±

of dead BMDMs (PI+) are shown within the dot plots. The statistically significant difference between the TSB 1 %G inoculum and the other inocula (P<0.01, ANOVA) is indicated by asterisks (**).

Fig. 5.

Effect of cell dormancy on peritoneal macrophage activation. Flow cytometric analysis of peritoneal cell exudates after intra-peritoneal challenge with 5×105 S. epidermidis bacteria obtained from biofilms grown in TSB 1 %G or TSB 1 %G+Ca2+/Mg2+. Dot plots are representative examples of two independent experiments (n = 4 per group). The proportions±

of non-activated macrophages (F4/80+Gr1−), activated macrophages (F4/80+Gr1+) or macrophages at a final stage of activation (F4/80−Gr1+) are shown within the dot plots. Cells with typical FSC and SSC parameters for lymphoid cells were excluded from the analysis. The statistically significant difference between the TSB 1 %G and TSB1 %G+Ca2+/Mg2+ groups (P<0.05, ANOVA) is indicated by an asterisk (*).

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Staphylococcus epidermidis biofilms with higher proportions of dormant bacteria induce a lower activation of murine macrophages - PubMed (original) (raw)