Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

doi:10.21203/rs.3.rs-115995/v1

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Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

https://doi.org/10.21203/rs.3.rs-115995/v1

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A major contributor to biomaterial associated infection (BAI) is Staphylococcus aureus. This pathogen produces a protective biofilm, making eradication difficult. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) comprising polysaccharides, proteins and extracellular DNA (eDNA). S. aureus also produces micrococcal nuclease (MN), an endonuclease which contributes to biofilm composition and dispersion, mainly expressed by nuc1. MN expression can be modulated by sub-minimum inhibitory concentrations of antimicrobials. We investigated the relation between the biofilm and MN expression and the impact of the application of antimicrobial pressure on this relation. Planktonic and biofilm cultures of three S. aureus strains, including a nuc1 deficient strain, were cultured under antimicrobial pressure. Results indicated that nuc1 deletion stimulates the polysaccharide production per CFU in the biofilm in in vitro biofilms. Also antimicrobial pressure of ciproflocacin, doxycycline and erythromycin resulted in significantly increased quantities of polysaccharides per CFU, but this did only coincide with significantly reduced MN activity in erythromycin. Resveratrol significantly reduced MN production per CFU but did not affect polysaccharides production. In conclusion, various antimicrobials impact the balance of eDNA, polysaccharides and MN production, all in a different way.

Applied & Industrial Microbiology

micrococcal nuclease

resveratrol

erythromycin

doxycyclin

sub-inhibitory

sub-MIC

Staphylococcus aureus

biofilm

An increasing number of biomedical implants are used in clinical practice. This is due to technical advancement, innovative therapies, increased patient demands and life expectancy. These implants come with the risk of biomaterial associated infection (BAI)¹. Simultaneously the rate of BAI per implant is increasing due to increasing age and comorbidities of the patients receiving such implants ^2,3. Treatment of BAI often entails revision of the implant and antibiotic therapy with a risk of secondary infection⁴. This results in increased morbidity and high healthcare costs¹. Primary antibiotic therapy is usually ineffective since bacteria create a protective shelter when attached to the surface of the foreign material, also designated as a ‘biofilm’. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) providing protection from antibiotics, the host immune system, and physical and mechanical stress⁵. The EPS content includes polysaccharides, proteins and extracellular DNA (eDNA), which plays a major role in biofilm maturation and structure^6,7. eDNA in particular has a glue-like function by keeping bacteria entangled within the biofilm by electrostatic and acid-base interactions with cell surfaces and polysaccharides^7,8.

One of the most prominent pathogens in BAI is Staphylococcus aureus, a strong biofilm producer responsible for 34% of all orthopedic BAI⁹. An important virulence factor of S. aureus is micrococcal nuclease (MN), a thermostable endonuclease that degrades eDNA as a constituent of the biofilm^10,11. Biofilm formation and maturation is expected to be intrinsically affected by the production of MN since it cleaves eDNA. MN production is largely regulated by the SaeRS gene system ^10,12. SaeRS consists of a sensory SaeS part and a SaeR response regulator and is activated mainly in the post-exponential growth phase by phagocytosis-related signals, sub-minimum inhibitory concentrations (sub-MIC) of some antibiotics and certain chemical stimuli¹³. S. aureus produces two types of MN. The first is secreted by the bacterium and encoded by the nuc1 gene. The second is cell-wall bound and encoded by nuc2^10,14. Nuc1 and nuc2 are expressed in different ratios depending on the growth phase, with nuc1 being expressed mainly in the post-exponential phase and nuc2 in the early-exponential phase^10,15. In post-exponential cultures of S. aureusnuc2 accounts for a minimal part of the DNA degrading capabilities of S. aureus cultures¹⁵. Our previous research estimated that about 1% of the nuclease activity at the post-exponential phase can be contributed to nuc2, as nuclease activity is decreased by 99% when comparing a S. aureus Newman and its nuc1 deficient mutant¹⁶. Controversy exists about the effect of MN on biofilm formation. A negative correlation was observed in vitro between biofilm biomass and the nuclease activity in biofilms of various S. aureus strains and nuc1 mutants¹⁷, whereas in an in vitro catheter model no effect was found on biofilm formation in a nuc1 and nuc2 mutants of S. aureus UAMS-1 strains¹⁸.

MN expression and in particular the SaeRS system can be modulated by sub-minimum inhibitory concentrations of antimicrobials (see also Table 1)^19,20. Sub-MIC antimicrobial pressure exists in tissue surrounding biomaterial implants because treatment by antimicrobials is hindered by poor penetration into the biofilm or by pathological changes in the implant site, like the formation of a fibrous layer around the implant in case of older implants (>1 month)²¹, or changes to the bone structure in joint prosthesis²².

This study aimed at investigating the relation between biofilm formation and MN production and the impact of sub-MIC antimicrobial pressure on this relation. Therefore biofilm formation and MN activity in planktonic and biofilm cultures were studied while applying various sub-MICs of antimicrobials to three strains of S. aureus, the S. aureus ATCC12600, the bioluminescent S. aureus Newman lux, and the S. aureus Newman lux Δnuc1 mutant which is deficient in producing MN¹⁷. The ATCC12600 is often used as a reference strain. The Newman WT strain was chosen because of its constitutive expression of MN due to a point mutation SaeS^p that constitutively activates the response regulator SaeR, even in the early-exponential growth phase²³. Five antibiotics were involved with different working mechanisms (Table 1) and one antimicrobial which has been shown to inhibit SaeRS activity¹⁹. In order to take into account biofilm characteristics that may be affected by the modulation of MN by sub-MICs of antimicrobials, relevant parameters in planktonic and biofilm cultures were investigated: the number of colony forming units (CFU’s) and bioluminescence as a measure of metabolic activity²⁴ and for biofilms total biomass and polysaccharide quantity in the EPS.

Table 1. Antimicrobials involved in this study, including working mechanism and effect on bacteria at sub-inhibitory concentration.

AntiMICROBIAL (group)	bacteriostatic/ bactericidal	bacterial substrate	sub-MIC effect
Ciprofloxacin (fluoroquinolone)	bactericidal	Inhibition DNA-gyrase	Increased expression alpha hemolysin and fibronectin binding protein^20,39
Doxycycline (tetracyclin)	bacteriostatic	Ribosomal 30S and 50S subunits	Inhibition endotoxins⁴⁰
Erythromycin (macrolide)	bacteriostatic	Ribosomal 50S subunit	Virulence reduction²⁰
Gentamicin (aminoglycoside)	bactericidal	Ribosomal 30S subunit	Virulence reduction²⁰ Poor biofim reduction⁴¹
Vancomycin (glycopeptide)	bactericidal	Inhibition cell wall synthesis	No effect on SaeRS¹³ Poor biofilm reduction⁴¹
Resveratrol (polyphenolic phytoalexin)	bacteriostatic	Affects tyrosine tRNA	Downregulation SaeRS¹⁹

CFU, polyaccharides and biomass of biofilms in absence of antimicrobials. The total biomass, polysaccharide content and CFUs of the two wildtype strain and one MN deficient S. aureus strain after 24 hours of biofilm growth were not significantly different (Fig. 1A-C). However, the biomass and polysaccharides per CFU of the MN deficient S. aureus Newman lux Δnuc1 grown under biofilm conditions were significantly higher than the other strains (Fig. 1D, E), indicating that an equally large biofilm was produced populated by fewer viable bacteria. Note that biomass and polysaccharide content do not need to be fully connected to each other, since biomass includes both bacteria (live and dead) and biofilm matrix.

MN production and cellular activity of biofilms and planktonic bacteria in absence of antimicrobials. Planktonically grown bacteria produced on average 49% more MN per CFU than those growing in a biofilm (P=0.04) (Fig. 2A). This was calculated in a mixed-effect analysis of lognormal MN/CFU values of the S. aureus ATCC12600 and Newman lux strains. As expected, the nuc1-deficient strain produced less than 1% of the amount MN produced by the parent strain. A mixed-effect analysis showed that the luminescence per CFU in strains grown planktonically overall was 150% higher than when grown in a biofilm (Fig. 2B). The luminescence was used as a marker for metabolic activity of the bacteria²⁴.

Figure 1. Biofilm characteristics of S. aureus ATCC12600, Newman lux, and Newman lux Δnuc1. (A) Biomass of biofilms measured by spectral absorption after crystal violet staining. (B) Polysaccharide content of biofilms measured by fluorescent intensity after calcofluor white staining (C) Colony forming units (CFU’s) per cm² of biofilms grown for 24 h in a 24 wells plate. (D) Ratios of biomass/CFU and (E) polysaccharides/CFU of biofilms. Data are normalized (S. aureus ATCC12600=1). All data shown are averages of triplicate measurements in three separate cultures, repeated on separate days. Error bars indicate standard error of the mean. * Indicates a significant difference (*: p<0,05; ***: p<0.001) calculated by T-test. In panel D the natural logarithm of the values was used for statistical analysis.

Figure 2. (A) Micrococcal nuclease (MN) per colony forming unit (CFU) of S. aureus ATCC12600 and S. aureus Newman lux from planktonic and biofilm cultures. The S. aureus Newman lux Δnuc1 mutant produced less than 1% of the amount of MN the non-deficient parent strain produced (data not shown). (B) Bacterial luminescence per CFU (photons/second/CFU) of S. aureus Newman lux and Δnuc1 mutant. Data shown are averages of triplicate measurements, repeated on separate days. Error bars indicate standard error of the mean. (***Indicates a significant difference: p<0.001).

Changes in biofilm composition under antimicrobial pressure As expected, most S. aureus biofilms required a higher concentration of antimicrobials to achieve inhibition of growth than planktonically grown bacteria resulting in MBIC > MIC (Table 2). The exceptions being doxycycline and erythromycin, for which no differences between planktonic and biofilm inhibition were observed for both Newman strains. It should be noted that the susceptibility to antimicrobial substances of the Newman nuc1-deficient strain was identical to its parent strain (Table 2).

Table 2. Resistance cutoff points ⁴², minimal inhibitory concentrations (MIC) and minimal biofilm inhibitory concentrations (MBIC)(µg/ml) of antimicrobials for Staphylococcus aureus ATCC12600, Newman lux, and Newman lux Δnuc1 strains.

		S. aureus ATCC12600		S. aureus Newman lux		S. aureus Newman lux Δnuc
	Resistant when MIC ≥	MIC	MBIC	MIC	MBIC	MIC	MBIC
Ciprofloxacin	4	1	2	8	16	8	16
Doxycycline	16	0.0625	0.5	0.125	0.125	0.125	0.125
Erythromycin	8	64	512	64	64	64	64
Gentamicin	16	2	8	1	2	1	2
Vancomycin	16	1	2	1	2	1	2
Resveratrol	Not used clinically	128	>512	>512	>512	>512	>512

Most sub-inhibitory concentrations of antimicrobials had no significant inhibiting effects on total biomass and polysaccharides (see Fig. 3). Ciprofloxacin had a stimulating effect on biomass at low doses (⅛ and ¼ MBIC) in both S. aureus Newman strains. Total MN activity of the biofilms was inhibited up to 80% by doxycycline, erythromycin and resveratrol at concentrations below the MBIC (See Fig. 3). At high concentrations (±100 μg/mL) resveratrol gradually precipitated, binding crystal violet and causing a strong false positive signal. Precipitated resveratrol, however, had no effect on the quantification of MN activity and polysaccharides (data not shown). No significant difference was found in biomass and polysaccharide between S. aureus Newman lux and the nuc1-deficient strain at any concentration of any antimicrobial (Fig. S1).

Effects of antimicrobials on EPS and nuclease activity per bacterium. CFU-counts in both planktonic and biofilm cultures (Fig. 4) were used to calculate the effect of antimicrobials on MN activity per viable bacterium. Under antimicrobial pressure of ciprofloxacin, doxycycline, and erythromycin, the biomass and polysaccharides per CFU increased significantly by up to five times that of the control (Fig. 5), whereas gentamicin, vancomycin and resveratrol had a moderate to no effect. This is due to the biomass and polysaccharide content staying largely unchanged while the number of CFU’s decreased (Fig 3 and 4). In contrast to the stimulating effect of some antimicrobials on biomass and polysaccharides, MN activity per CFU was unaffected or inhibited by antimicrobials (Fig. 6). In particular resveratrol inhibited MN per CFU significantly at concentrations as low as ⅛ MIC and MBIC, except for S. aureus ATCC12600 biofilms in which MN per CFU was inhibited at ⅛ and ¼, but not at ½ MBIC. Erythromycin reduced MN activity of the Newman strain by almost 75% at ¼ MBIC.

The aim of this study was to investigate the relation between biofilm formation and MN activity and the impact on it by sub-MIC antimicrobial pressure. Various earlier reports are not unambiguous on whether MN has an inhibitory effect on biofilm formation with results varying from increased to decreased biofilm formation, also depending on in vitro¹⁷ or in vivo setup ^11,17,18,25. Our results (Fig. 1D,E) revealed no significant differences between the two WT-strains and the MN-deficient strain with respect to the number of CFU, the total biomass and polysaccharides (fig. 1 A-C). There are several possible explanations for the observation that biofilms in our experiments did not increase in mass upon deletion of the nuc1 gene as was observed in other research¹⁷, such as the lack of added glucose in the growth medium, longer incubation time (24 hours), and the lower initial bacterial load that may alter the response of the biofilm to MN ^11,26,27.

When the present results, however, were normalized with respect to the number of CFU’s in the assays, the results revealed, for the first time, a significant, 20% enhancement of biomass and polysaccharides production per CFU of the nuc1 deficient S. aureus Newman lux strain compared to both other S. aureus strains. This suggests an inverse relationship between EPS production per CFU and MN activity per CFU. This is in line with earlier findings²⁵, based on bacterial counts and optical coherence tomography observations, that the bacterial density in biofilms is reduced in the nuclease deficient S. aureus Newman strain. Uncleaved eDNA in the nuclease deficient strain tends to expand the biofilm and may keep bacteria at relatively long distances from each other, also pointing to an increased biomass production per CFU in MN deficient strains²⁵.

In this study we anticipated that some antibiotics may modulate MN production. Significant reductions were observed for doxycyclin, erythromycin and resveratrol by up to 80% (Fig. 3). The inhibition of MN activity by erythromycin is in line with reports of clindamycin, which has a similar working mechanism by binding to the ribosomal 50s subunit and inhibits the SaeRS system (Table 1). The reduction by resveratrol is attributed to the direct inhibition of the SaeRS system as reported earlier¹⁹. Since it was concluded from Fig. 1E that MN reduction had effect on the EPS production per CFU rather than on the total CFU count (Fig. 1C), biomass (Fig. 1A) and polysaccharides (Fig. 1B), it was expected that EPS production per CFU subsequently would be affected by sub-MIC pressure of antimicrobials as well. Indeed significant higher polysaccharide content per CFU was found in the presence of sub-MIC doxycyclin and erythromycin, up to a factor of 5 with respect to the control (Fig. 5), but only in case of erythromycin this coincides with significant reduction in MN per CFU (Fig. 6) and not in case of sub-MIC doxycyclin. These results strongly suggest that high polysaccharide production in the presence of sub-MIC antimicrobials are not only a result of reduced MN activity.

It is more likely that sub-MIC antibiotics impact not only the SaeRS regulatory system but also other genetic systems that have an effect on the biofilm, such as potentially the icaA system expressing polysaccharides²⁸ and the AtlA autolysin gene responsible for programmed cell lysis and eDNA release²⁹. It was found earlier in S. aureus that in particular AtlA is directly or indirectly regulated by the SaeRS two component system³⁰. Antibiotics, having effect on the combined expression of Atla, SaeRS and icaA , may thus disturb this delicate balanced expression of nuclease and polysaccharides and release of e-DNA in various ways. This is illustrated by biofilms incubated with sub-MIC erythromycin, which significantly affected nuclease production and stimulated polysaccharide production, but failed to increase biomass.

MN activity was significantly inhibited in the biofilm mode of growth compared to planktonic growth (Fig. 2A). The reduction of MN in biofilms compared to planktonic cultures, however, is similar to the reduction of bioluminescence in biofilms as related to planktonic cultures. The reduction in bioluminescence most likely results from a limited availability of oxygen and lower metabolic activity of bacteria located deeper in the biofilm ^24,31. Decreased oxygenation and metabolic activity is probably also the dominant factor reducing MN production in biofilms.

In summary our results do not confirm earlier findings that MN directly influences total biomass of the biofilm, but rather that deletion of nuc1 stimulates the EPS production per CFU. MN production can be modulated by antimicrobial pressure, and did in some cases coincide with significantly increased production of polysaccharides, but this was not a consistent relationship and might be a result of other more complex regulatory mechanisms.

As stated previously, sub-MIC antibiotic pressure develops around biomaterial implants during antibiotic treatment. Therefore, sub-MIC antibiotics or antimicrobials are relevant with regard to BAI. Modulation of MN production of S. aureus may result in clinically relevant strategies of preventing and treating S. aureus infections by arresting biofilm dispersal. Moreover S. aureus bacteria possess escape mechanisms from the host immune system in particular by MN that disassembles neutrophil extracellular traps, rendering the bacteria unaffected by this particular defense mechanism of the immune system^32,33. Reduction of MN by sub-MIC antimicrobials may thus assist in the eradication of biofilms by the host immune system, even in case of clinical resistance to these antimicrobials.

Antimicrobials. The antimicrobials used are mentioned in Table 1 together with their working mechanism. They were purchased from Merck (Darmstadt, Germany). All antimicrobials were prepared from powder-form in ultrapure water and sterilized by filtering through a 0.22 μm pore filter according to the manufacturers protocol. Resveratrol was dissolved in DMSO (dimethylsulfoxide) to a concentration of 52 mg/ml before filtration. Due to the poor aqueous solubility of resveratrol no concentrations of resveratrol higher than 512 µg/ml in ultrapure water containing 1% DMSO were used. All cultures containing resveratrol including the control for these cultures without antibiotics, contain 1% DMSO.

Nuclease probe. In order to measure MN production a nuclease activatable fluorescence probe was applied ^16,34. The probe (Merck) consists of a 11-thymine base oligonucleotide flanked by a fluorescein amidine fluorophore and both a ZEN and Black hole quencher (5’-/FAM/TTTTTTTTTTT/ZEN/BhQsp/-3’). Before use the lyophilized probe was diluted in 10 mM Tris-HCl and 10 mM CaCl₂, pH 8.0 to a working stock concentration of 2 µM.

Bacterial strains, culturing and harvesting. Experiments were conducted with S. aureus ATCC12600 and a luminescent S. aureus Newman lux strain (AH2600) in which LuxABCDE genes and kanamycin resistance were transduced from Photorhabdusluminescens using bacteriophage 11 ³⁵. The bioluminescence is the result of a LuxABCDE gene, regarded as a global marker for cellular activity. The S. aureus Newman lux Δnuc1 mutant strain (AH2627) was obtained by deletion of nuc1 using the Targetron Gene Knockout System (Merck) ¹⁷. Both S. aureus Newman strains were constructed previously ^17,34.

All media were prepared according to manufacturer’s protocol. S. aureus ATCC12600, S. aureus Newman lux and S. aureus Newman lux Δnuc1 were cultured from cryopreservative beads onto Tryptic Soy Broth Agar (TSA) (Oxoid, Basingstoke, UK). Kanamycin (200 μg/ml) was added to the agar plates, pre-, and main-cultures of the S. aureus Newman lux strains. After inoculation the agar plates were incubated for 24 h at 37ᵒC in ambient air.

A pre-culture was made by inoculating one colony in tryptic soy broth (TSB) (10 ml) (Oxoid) and cultured for 24 h at 37ᵒC, 150 RPM. The main culture was made by inoculating 40 ml TSB with 2 ml of the pre-culture and cultured for 16 h at 37°C, 150 RPM.

The bacteria were harvested by centrifugation for 5 min at 10°C, 5000 g (Avanti J-E centrifuge, JLA-16.259 rotor, Beckman-Coulter) and subsequently washed three times with phosphate buffered saline (PBS). All cultures were sonicated three times for 10 s at 30 Watt on ice, to remove aggregates. The number of bacteria was determined from a 1:200 diluted sample in a Bürker-Türk counting chamber in order to establish the required concentration of bacteria to start the experiments.

Minimum inhibitory concentration and minimum biofilm inhibitory concentration. The MIC of all antimicrobials were determined by incubating all precultured strains for 24 h at 37 °C in a 1:1 serially diluted antimicrobial concentration starting from 512 µg/ml. To this end all wells but the first were filled with 100 μl of ultrapure water. Then an antimicrobial solution (200 μl) at double the final concentration (1024 µg/ml in this case) was added to the first well. Subsequently 100 μl was transferred from the first well to the next, and then from that one to the next etc. Then a suspension of double the final concentration of bacteria (2*10⁵ cells/mL in this case) in double concentrated TSB was prepared, and 100 μl was added to all wells, resulting in all wells containing the final concentration of bacterial inoculum in TSB, with an antimicrobial gradient. Wells with sterile TSB were included as negative controls. After 24 hours the plates were assessed for the lowest concentration that resulted in no visible growth (MIC).

To determine the minimum biofilm inhibitory concentration (MBIC), wells in a flat-bottom 96 wells plate (Greiner Bio-One) were filled with 200 μl of 5*10⁸ bacteria/ml in TSB. Bacteria were allowed to adhere for 1 h at 37 °C under stationary conditions. Non-adhering bacteria were removed by washing three times with PBS before addition of an antimicrobial gradient as described above, followed by stationary incubation for 24 h at 37 °C. Biomass was then assessed by staining with crystal violet and spectral absorption. To assess the biomass, the biofilms were washed gently three times with PBS and dried at 60°C to fix the biofilms. Biofilms were stained for 5 min with crystal violet (0.06%) and washed 3 times with demineralized water ³⁶. Subsequently, crystal violet was resolubilized in 30% acetic acid for at least 15 min. Finally, the crystal violet solution was diluted 4 times and the absorbance was measured at 590 nm in a Fluostar Optima plate reader (BMG labtech, Ortenberg, Germany). This device is able to measure absorbance up to a value of 4.5. Values measured across all experiments did not exceed 50% of this maximum. MBIC was defined as a ≥90% inhibition of biofilm formation ³⁷.

Effect of antimicrobials on planktonic cultures. All planktonic cultures were grown in a 24 (polystyrene) well plate (Greiner Bio-One, Frickenhausen, Germany) with a final volume of 1120 μl per well with decreasing antimicrobial concentrations, established by a 1:1 serial dilution ranging from 4 times to 1/16 times the MIC as described above with adjusted volumes. Wells with sterile TSB were included as negative controls. The final volume of 1120 μl for each well was chosen to preserve the volume-to-surface ratio across all experiments. 1% DMSO was added to the wells that served as a control for the strains grown with resveratrol. The concentration of bacteria at T=0 was 1*10⁵ bacteria/ml. All cultures were grown as described under MIC and MBIC.

For each of the concentrations of antimicrobials the number of CFU’s and the production of MN were determined in the same bacterial culture in order to allow the calculation of MN production per CFU.

Effect of antimicrobials on biofilm cultures. Biofilms were grown as described above. The differential concentrations of antibiotics were established by a 1:1 serial dilution that ranged from 1 time to 1/8 times MBIC. 1% of DMSO was added to the wells that served as a control for the strains grown with resveratrol. Wells with sterile TSB were included as negative controls. Separate biofilm cultures were prepared for measuring biofilm biomass, polysaccharide quantification, and CFU count/MN production. The former two were quantified from a single biofilm culture in order to allow the calculation of nuclease production per CFU. In order to relate biofilm formation at various antimicrobial concentrations, biofilms of all strains were grown simultaneously on the same day for all concentrations.

Determination of biomass and polysaccharides. To assess the biomass of the biofilm the same crystal violet procedure was followed as described for the determination of the MIC and MBIC. To quantify the polysaccharides 100 μl medium was carefully removed from each well containing a biofilm and replaced by 100 μl calcofluor white (Sigma Aldrich) solution (40 μg/ml) to yield a final concentration of 20 μg/ml calcofluor white per well which binds to polysaccharides ³⁶. After 1 min all wells were gently washed three times with PBS. Biofilms were resuspended by pipetting forcefully up and down and 5 min of sonication of the plate in a sonication bath. Fluorescence was measured in a Fluostar Optima plate reader (Excitation: 355 nm/ Emission: 490 nm, setting: bottom).

Determination of CFU’s. All biofilms were resuspended by pipetting forcefully up and down and 5 min of sonication of the plate in a sonication bath. From the 24-h planktonic cultures a sample was taken directly from the suspension. The samples (20 μl) were serially tenfold diluted in PBS (180 μl). Three 10 μl aliquots of every dilution (10-10⁷ times diluted) were put on a TSA plate and grown for 18 h at 37°C. Then the number of colonies were counted, and the number of CFU per cm² (biofilms) or per mL (planktonic) was calculated.

Nuclease activity. Bacterial suspensions of resuspended biofilm and planktonic cultures were obtained from the same cultures as from which the samples for CFU counting were taken. To measure nuclease activity samples were diluted 1000 times with 10 mM Tris-HCl, 10 mM CaCl_2,pH 8.0 buffer. Twenty-five µl of diluted bacterial suspension was combined with 25 µl of probe working stock (2 µM) and 10 mM Tris-HCl, 10 mM CaCl_2,pH 8.0 (150 µl), buffer in a 96 wells plate. As a negative control 1:1000 diluted sterile TSB was used instead of a sample from the culture. Fluorescence intensity was measured with a Fluostar Optima plate reader (Excitation: 490 nm; Emission: 520 nm) at 1-min intervals for 5 min at 37 °C. Nuclease activity was determined by the rate of fluorescence change per min. We used a known amount of purified MN (Merck) to calibrate the nuclease probe. The rate of fluorescence per min was shown to be linear with MN concentration (Fig. S2). One unit (U) is defined as the amount of enzyme required to release acid soluble oligonucleotides that produce an absorbance increase of O.D. 1.0 at 260 nm in 30 min at 37°C, pH 8.8 ³⁸.

Bioluminescence. To quantify bioluminescence, plates containing the biofilm or planktonic cultures were placed in an IVIS Lumina 2 system (PerkinElmer, Waltham Massachusetts US) and imaged for one minute (excitation filter: blocked, emission filter: open). Data was analyzed using the LivingImages 4.7.2 software (PerkinElmer). Reported units are in photons per second leaving the entire volume of the well.

Statistical analysis. All data were analyzed using Graphpad Prism 8 (Graphpad, San Diego, United States). Difference between 2 groups was calculated using a T-test, unless stated otherwise. All statistics were based on log-normal values (Fig. S3). Differences between lognormally distributed data were calculated using the logarithmic values. All experiments were performed in triplicate on each of three separate days with different cultures. Reported values are average values over nine measurements, standard deviations, standard errors of the mean and statistical tests are based on the three average values obtained, one for each culture.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

This project received funding from the UMCG. The authors express their gratitude to Prof. McNamara for providing the S. aureus Newman strains utilised in this study.

Author contributions

H.C.V.M., J.S. and C.W.K.R. planned the study and wrote the manuscript. C.W.K.R. performed the experiments, analysed the data and prepared the figures. All authors reviewed the manuscript.

competing interests

The authors declare no competing interests.

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Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

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