Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.


Low doses of paracetamol increase biofilm formation of CC8 genetic cluster strains.
To study the effect of paracetamol on biofilm formation, a biofilm quantification assay was performed on all strains following overnight incubation with paracetamol using the same concentrations as used in the planktonic growth experiments. In 15 of the 21 strains, an increase in biofilm mass could be demonstrated (Fig. 2). Of particular note, this phenomenon was primarily observed in S. aureus strains belonging to CC8 (2774 (ST72), M116 (ST239), RWW337 (ST239), RWW338 (ST239), SAC042W (USA300), 8325-4, RN6390, and Newman), and less visible in non-CC8 strains (CC1 (903), CC5 (2778, 3904), CC15 (1675), CC30 (2091, 3534), and CC223) when exposed to doses of paracetamol less than 32 µg/mL. Not every paracetamol concentration below 32 µg/mL always led to a clear increase in biofilm mass. For example, the mean value of strain M116 (ST239) indicates biofilm mass increased at 0.5-4 μg/mL and 16 μg/mL, whereas at 8 μg/mL biofilm mass was unaffected. Conversely at these doses, other strains of various backgrounds were observed with mainly reductions in biofilm and as low as obtaining only 63% (mean) of the control biofilm mass for 8 µg/mL paracetamol (strain 3648 (CC1); Fig. 2E).
Paracetamol did not significantly alter the metabolism rate of biofilm-associated S. aureus. To determine if the paracetamol-associated increase in biofilm formation was only due to an increase in the number of bacterial cells, we studied the metabolic rate of S. aureus biofilms (CC20 strain M82 and CC8 strains: M116, RN6390, and Newman) when exposed to different concentrations of paracetamol. The addition of paracetamol did not change the heat flow by S. aureus during 24 h biofilm formation for all strains tested (Fig. 3). Some minor, non-significant deviations included a delayed heat curve at 4 µg/mL in strain M82 (CC20) (Fig. 3A) and reduced heat flow at the highest therapeutic concentration (32 µg/mL) in strain RN6390 (CC8) (Fig. 3C). These data indicate that although the biomass of biofilms of CC8 strains generally increased when exposed to paracetamol, an increase of bacterial cells is unlikely to be the cause.
Paracetamol modulates polysaccharide expression during biofilm formation. An explanation for the increase of biofilm mass could be an increase in non-cellular components, such as extracellular DNA (eDNA), proteins and/or polysaccharides 1,27-31 . To investigate this hypothesis, we co-incubated S. aureus strain M116 (CC8) biofilms with 2 μg/mL of paracetamol overnight, and studied the biofilms using light fluorescence and confocal microscopy (Fig. 4). Paracetamol-exposed biofilms had an increase of N-Acetyl glucosamine content in their extracellular matrix (Fig. 4).
Small doses of paracetamol increase immune-modulator transcription. RN6390 (CC8) containing a SCIN promoter-green fluorescence protein fusion was used to study the effect of paracetamol exposure on the transcription of the immune modulator SCIN protein. During overnight co-incubation with paracetamol, the biofilms had a significant upregulation of scn promoter transcription (p = 0.009) when lower doses (< 4 µg/ mL) of paracetamol were added during biofilm formation ( Fig. 5A-C). No difference was observed with a higher dose of 4 µg/mL (Fig. 5D).

Discussion
In this study, we show that paracetamol exposure can lead to an increase in biofilm mass of S. aureus strains, particularly from the CC8 genetic background which includes the highly prevalent ST239 and USA300 strains. Bacterial metabolism during biofilm formation did not significantly increase as a consequence of adding paracetamol. It could be that the total number of biofilm-associated bacteria exposed to paracetamol is less than the untreated bacteria, but those fewer exposed cells are producing more heat due to the increased production of www.nature.com/scientificreports/ polysaccharides and secreted proteins. However, the heat production due to polysaccharide and secreted protein generation might be negligible in comparison to the heat produced due to cellular growth. Regardless, in both cases there is no increase in the number of biofilm-associated bacterial due to paracetamol treatment and thus, the observed increases of biofilm mass due to paracetamol exposure seems to be best explained by an increase of non-cellular components. Furthermore, the increase in biofilm mass was associated with an increase in the part of the biofilm rich in N-Acetyl glucosamine, presumably polysaccharide intercellular adhesin (PIA). PIA is a positively charged poly-β(1-6)-N-acetylglucosamine (PNAG) and the predominant exopolysaccharide component of the extracellular polymeric matrix of staphylococcal biofilms encapsulating bacteria 30,32 . Additionally, PIA mechanically and chemically prevents the killing of S. aureus by phagocytizing leukocytes, antimicrobial peptides, and antibiotics 30,32,33 . No previous data regarding PIA modulation by paracetamol exists to our knowledge. However, a study by Dotto,et al. 18 showed that acetylsalicylic acid promotes biofilm formation of S. aureus in a PIAdependent manner. These data suggest that use of both paracetamol and acetylsalicylic acid during S. aureus infections may be potentially harmful due to increasing biofilm formation.
Bacterial secreted proteins were another consideration in our study. Since the regulator pathway for biofilm formation and toxin production, including immune modulators, are correlated 23,24 , we expected to find that www.nature.com/scientificreports/ immune modulator production by S. aureus could be affected by paracetamol as well. Our data indeed showed that SCIN production can be stimulated significantly by low doses of paracetamol. This finding mimics the data from Price et al. 34 , which demonstrated that acetylsalicylic acid could modulate S. aureus virulence factor production. SCIN is a potent immune modulator, which is able to inhibit the release of chemoattractant C5a and therefore blocking the host complement activation pathways 35,36 . Previously, we showed that S. aureus produced SCIN already during the early stages of biofilm formation 12 . The observed increase in SCIN production, along with PIA and potentially other virulence factors yet to be investigated, in response to paracetamol would aid invading staphylococci to evade the innate immune system and potentiate infection. Currently, the mechanism of biofilm modulation by paracetamol has not been elucidated, but there are indications that an impaired iron regulation within cells may influence this phenomenon 37,38 , probably via iron chelation by paracetamol 39 . In an iron-restricted condition, biofilm and virulence factor production is increased 18,40 . Paracetamol has been demonstrated in vivo to reduce excess hepatic iron after administration 41 . In addition, a previous study on acetylsalicylic acid and biofilm showed that free Fe 2+ reduction in culture media by acetylsalicylic acid, via iron chelation, could promote biofilm formation of S. aureus CC5 and CC8 strains, including Newman and USA300 18 . This observation suggests that iron-modulation by paracetamol may enhance S. aureus biofilm formation. CC15 (1675) CC15 (568) CC5 (3904) CC5 (2778) CC1 (3648) CC1 (903) Biofilm mass relative to control (%) Biofilm mass relative to control (%) www.nature.com/scientificreports/ In summary, this study indicates that current clinical concentrations of an analgesic-antipyretic like paracetamol may have a role in the development and persistence of S. aureus biofilm-related infections, especially, but not limited to, strains belonging to CC8. For clinical practice our data suggest that in patients with a suspected S. aureus infection, the indication for paracetamol administration should be carefully weighed against the risk of increased biofilm formation. The mechanism of action and the effect on an established, mature biofilm by paracetamol need to be investigated in future studies.

Materials and methods
Bacterial strains and growth condition. The S. aureus strains used in this study are listed in Table 1.
Biofilm formation mass assessment. In vitro biofilms were generated from both wild type and GFPcontaining strains (Table 1) by the method described previously 12 . Biofilm formation was assessed using a dynamic, microtiter plate biofilm formation assay as previously described by Christensen et al. 2,12,43,44 , with slight modifications. Briefly, for each strain or isolate, 5 mL of NaCl 0.9% was inoculated with overnight grown S. aureus (at 37 °C on blood agar) until an OD 600nm of 0.50 (± 0.05) was reached. Thereafter, 10 µL was dispensed    Bacterial metabolism rate measurement. The effect of paracetamol on bacterial metabolism was monitored with a microcalorimeter according to the previously described protocol 11 . Briefly, overnight cultures of S. aureus on blood agar at 37 °C were suspended in 5 mL NaCl 0.9% until OD 600nm of 0.50 (± 0.5) was reached. Then 10 µL of this suspension was mixed with 9990 µL IMDM to create a 1:1000 dilution. Ten µL of the diluted suspension was added into sterile plastic "insert" tubes (designed for the microcalorimeter) containing 190 µL of different concentrations (0.5-32 mg/mL) of paracetamol in IMDM. These tubes were then placed into sealed

Measurement of immune modulators SCIN transcription.
To study the effect of paracetamol on the transcription of the immune modulator SCIN during biofilm formation, RN6390 carrying a scn (SCIN) promotor GFP construct and RN6390 carrying a plasmid that constitutively produces GFP as a positive control (phage repressor promoter-GFP fusion construct) were used as previously described by Rooijakkers et al. 47 ( Table 1). In vitro biofilms were generated from both GFP-containing strains (Table 1) by the method described previously 12 . After an hour of incubation at 37 °C, the growth medium was replaced with 200 µL of fresh IMDM medium containing paracetamol (serial dilution 0.5-32 μg/µL). The biofilms were then incubated in a FLUOstar Optima microplate fluorescence reader (BMG Lab Technologies, Chicago, IL, USA) at 37 °C with 150 rpm periodic rotational shaking. The accumulation of fluorescence, used as a measure for gene transcription, was determined (excitation at 485 nm, emission at 520 nm, and gain setting 1738) automatically every 5 min during the 4th and 5th hours of biofilm formation. Median fluorescence intensities (MFI) of the scn promoter-GFP strain co-incubated with paracetamol were compared to the MFI of the strain incubated without paracetamol.

Data analysis.
Confocal image acquisition and analysis were obtained using Nikon Instruments Software (NIS)-Elements Advanced Research (Version 4.50; Nikon Instruments). Statistical analysis and data graphical representation was performed with GraphPad Prism (GraphPad Software Inc., Version 5.01 for Windows, San Diego, CA, USA, www.graph pad.com) and Microsoft Excel (Microsoft Corporation, 2010, https ://offic e.micro soft.com/excel ). Data were analyzed with an unpaired t-test or one-way ANOVA; a P-value ≤ 0.05 was considered as statistically significant. All experiments were independently repeated at least three times and data are presented as mean (SEM) or as median with range.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. scn-GFP ( +) CC8 scn promoter (pSK236GFP in RN6390) 54 PrP-GFP ( +) CC8 Page repressor promoter (pACL1484 in RN6390) 47