scholarly journals Structural and Functional Dynamics of Staphylococcus aureus Biofilms and Biofilm Matrix Proteins on Different Clinical Materials

2019 ◽  
Vol 7 (12) ◽  
pp. 584 ◽  
Author(s):  
Anna K. Hiltunen ◽  
Kirsi Savijoki ◽  
Tuula A. Nyman ◽  
Ilkka Miettinen ◽  
Petri Ihalainen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Matrix Proteins ◽  
Clear Correlation ◽  
Detailed Knowledge ◽  
Time Points ◽  
Functional Dynamics ◽  
Associated Proteins ◽  
Biofilm Development ◽  
Surface Adhesins

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials—borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene—at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points—from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points.

scholarly journals Interaction of Salmonella with E. coli and Proteus spp. in Biofilm Formation

2021 ◽  
pp. 30-45
Author(s):  
S. U. Pathiranage ◽  
D. N. N. Madushanka ◽  
K. V. D. M. Hasintha ◽  
H. C. Nadishani ◽  
G. C. P. Fernando ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Broiler Chicken ◽  
Chicken Meat ◽  
Salmonella Spp ◽  
E Coli ◽  
Time Points ◽  
Dual Cultures ◽  
Microtitre Plate Assay ◽  
Biofilm Development ◽  
Numerous Time

Aims: Investigate the interaction of Salmonella spp. with E. coli and Proteus spp. in biofilm formation as mono and dual-species at different time durations Experimental Design: Salmonella, Proteus, and E. coli were isolated from Broiler chicken meat, and the biofilm-forming ability of these organisms were studied. Place and Duration of Study: The study was conducted at the Laboratory of Livestock Production, Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, from 2019 December to 2020 May. Methodology: This study investigated the biofilm-forming ability of Salmonella as a mono species and its interaction with E. coli and Proteus in the process of biofilm formation. Microorganisms used for this study were isolated from broiler chicken meat. Biofilm was quantified using a microtitre plate assay. The interaction effects were tested at the temperature of 280C in different time durations (up to 120 hours). Results: Salmonella 1 and Proteus monocultures showed significantly higher biofilm-forming ability than Salmonella 3 isolate at all tested time points. At 120 hr, additionally to the salmonella 1 and Proteus isolates E. coli also formed significantly higher biofilms than Salmonella 3. However, Salmonella 3 was the lowest biofilm former as mono biofilm at all tested time durations. Salmonella 1 interaction with Salmonella 3 isolates formed less biofilms than Salmonella 1 mono biofilm at 48hr and 72hr correspondingly. Salmonella 1 and its interactions with Salmonella 3, Proteus, E. coli showed similar biofilm-forming abilities without significant differences at all other tested time points. Specifically, Salmonella 3 interaction with Salmonella 1 as dual biofilm showed higher biofilm-forming ability than Salmonella 3 mono biofilm at all tested time points. Tested isolates and their interaction achieved the highest biofilm formation at numerous time points. In fact, at 48hr, Salmonella 3 isolates and its interaction of Proteus, E. coli, and Salmonella 1 interaction with Proteus attained their highest biofilm formation abilities. The highest biofilm formation was achieved by Salmonella 1 isolate as mono biofilm and Salmonella 1 interaction with E. coli as dual biofilm at 72hr. Biofilm-forming trend of respective isolates and interactions showed numerous patterns at tested time durations. Specifically, E. coli rapidly enhanced its biofilm-forming ability as monoculture from 24 hr to 120 hr. Proteus, Salmonella 3 as monocultures, Salmonella 3 interaction with Proteus and E. coli as dual cultures showed progressive biofilm development from 24 hr to 48 hr. Salmonella 1 monoculture and its interaction with Salmonella 3, E. coli as dual biofilm improved their biofilm-forming ability from 24 hr to 72 hr. Similar to Salmonella 3 interaction with Proteus, Salmonella 1 interaction with Proteus also increased its biofilm-forming ability from 24 hr to 48 hr. Conclusions: This study concluded that there is a variation among isolates and their combinations in forming the biofilms, where there is an enhancement of biofilm in dual-species over the mono-species in some interaction, and there is a reduction in biofilm formation by dual-species with some combinations. Further, this concluded that Salmonella is interacting with other commonly found bacteria such as Proteus and E. coli in biofilm formation.

scholarly journals Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

2008 ◽  
Vol 191 (3) ◽  
pp. 832-843 ◽  
Author(s):  
Nekane Merino ◽  
Alejandro Toledo-Arana ◽  
Marta Vergara-Irigaray ◽  
Jaione Valle ◽  
Cristina Solano ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Protein A ◽  
Surface Proteins ◽  
Growth Media ◽  
Dependent Manner ◽  
Chronic Infections ◽  
The Matrix ◽  
Implanted Medical Devices ◽  
Biofilm Development

ABSTRACT The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

scholarly journals Inhibitory effect of a natural phenolic compound, 3-p-trans-coumaroyl-2-hydroxyquinic acid against the attachment phase of biofilm formation of Staphylococcus aureus through targeting sortase A

RSC Advances ◽  
2019 ◽  
Vol 9 (56) ◽  
pp. 32453-32461
Author(s):  
Yan-Ping Wu ◽  
Xiao-Yan Liu ◽  
Jin-Rong Bai ◽  
Hong-Chen Xie ◽  
Si-Liang Ye ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Phenolic Compound ◽  
Biofilm Formation ◽  
Inhibitory Effect ◽  
Sortase A ◽  
Initial Attachment ◽  
Biofilm Development

3-p-trans-Coumaroyl-2-hydroxyquinic acid (CHQA), a natural phenolic compound, prevented Staphylococcus aureus biofilm formation due to the inhibition of the initial attachment stage of biofilm development by targeting sortase A.

scholarly journals Antibiotic susceptibility of Staphylococcus aureus with different degrees of biofilm formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hyo-Jung Shin ◽  
Sungtae Yang ◽  
Yong Lim
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Resistance Mechanisms ◽  
Minimal Bactericidal Concentration ◽  
Chronic Infections ◽  
Planktonic Bacteria ◽  
Growth Modes ◽  
Biofilm Bacteria ◽  
Biofilm Development

AbstractStaphylococcus aureus is one of the most common pathogens in biofilm-associated chronic infections. S. aureus living within biofilms evades the host immune response and is more resistant to antibiotics than planktonic bacteria. In this study, we generated S. aureus with low and high levels of biofilm formation using the rbf (regulator of biofilm formation) gene and performed a BioTimer assay to determine the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of various types of antibiotics. We showed that biofilm formation by S. aureus had a greater effect on MBC than MIC, probably due to the different growth modes between planktonic and biofilm bacteria. Importantly, we found that the MBC for biofilm S. aureus was much higher than that for planktonic cells, but there was little difference in MBC between low and high levels of biofilm formation. These results suggest that once the biofilm is formed, the bactericidal activity of antibiotics is significantly reduced, regardless of the degree of S. aureus biofilm formation. We propose that S. aureus strains with varying degrees of biofilm formation may be useful for evaluating the anti-biofilm activity of antimicrobial agents and understanding antibiotic resistance mechanisms by biofilm development.

scholarly journals Understanding the Role of the Antioxidant Drug Erdosteine and Its Active Metabolite on Staphylococcus aureus Methicillin Resistant Biofilm Formation

Antioxidants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1922
Author(s):  
Cristina Cattò ◽  
Federica Villa ◽  
Francesca Cappitelli
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Active Metabolite ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Major Effect ◽  
Total Proteins ◽  
Methicillin Resistant ◽  
Biofilm Development ◽  
Open Question

Increasing numbers of researches have suggested that some drugs with reactive oxygen species (ROS)-mediated mechanisms of action modulate biofilm formation of some pathogenic strains. However, the full contribution of ROS to biofilm development is still an open question. In this paper, the correlations between the antioxidant drug Erdosteine (Er) and its active Metabolite I (Met I), ROS and biofilm development of two strains of methicillin resistant Staphylococcus aureus are presented. Experiments revealed that Er and Met I at 2 and 5 mg/L increased up to three orders of magnitude the number of biofilm-dwelling cells, while the content of ROS within the biofilms was reduced above the 87%, with a major effect of Met I in comparison to Er. Comparative proteomics showed that, 5 mg/L Met I modified the expression of 30% and 65% of total proteins in the two strains respectively. Some proteins involved in cell replication were upregulated, and a nitric oxide-based mechanism is assumed to modulate the biofilm development by changing quorum sensitive pathways. Additionally, several proteins involved in virulence were downregulated in the presence of Met I, suggesting that treated cells, despite being greater in number, might have lost part of their virulence.

The effects of NX-AS-401 on methicillin resistant Staphylococcus aureus

2021 ◽  
Author(s):  
◽  
Phillip Butterick
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Galleria Mellonella ◽  
Standard Of Care ◽  
Health Concern ◽  
Current Standard ◽  
Methicillin Resistant ◽  
Biofilm Development ◽  
Strain Dependent

Antimicrobial resistance is a global health concern, with once treatable infections becoming resistant to current standard of care antimicrobials. The search for new antimicrobials has led Neem Biotech Ltd. to manufacture NX-AS-401 an ajoene containing compound derived from Allium sativuum, commonly known as garlic. The research contained within this thesis aimed to identify the effects of NX-AS-401 on Methicillin Resistant Staphylococcus aureus (MRSA), one of the most well documented and commonly isolated antimicrobial resistant bacterial pathogens. A multi-stage approach was utilised, identifying how NX-AS-401 affects planktonic growth, biofilm development and virulence factor production. In Chapters 3 and 4 initial comparison between different NX-AS-401 formulations was performed in determined that ajoene content did not alter the antimicrobial effect of NX-AS-401. EUCAST broth microdilution compared NX-AS-401 to current standard of care antibiotic and determined effective inhibitory and bactericidal concentrations as 128 µg/ml and 2048 µg/ml respectively. When NX-AS-401 was used in combination with various antibiotic classes a synergistic effect was identified and the inhibitory concentrations of both agents were reduced. The primary focus on Chapter 5 was how NX-AS-401 affected S. aureus biofilm formation. NX-AS-401 concentrations of 32 µg/ml inhibited biofilm formation and a concentration of 512 µg/ml caused disruption of pre-established biofilms. These effects were confirmed using scanning electron microscopy and confocal microscopy with live/dead staining. In gene expression studies it was determined that the effects of NX-AS-401 on S. aureus biofilms were strain dependent and a target gene was not identified. Chapter 6 demonstrated that NX-AS-401 did not alter the production of Staphylococcus aureus exo-enzyme production in vitro during phenotypic studies. In Galleria mellonella low NX-AS-401 concentrations assisted in the recovery from S. aureus in a strain dependent manner, however, high concentrations caused increased Galleria mellonella fatality. NX-AS-401 altered the ability of S. aureus cells to invade human epithelial cells but did not prevent adhesion of S. aureus to the cells. NX-AS-401 has multiple effects on S. aureus with the ability to affect both planktonic cells and biofilm structure showing promise as an antimicrobial. Its main effects are growth inhibition and biofilm disruption rather than causing bacterial cell death. These attributes and the synergistic effects between NX-AS-401 and multiple antibiotic classes, indicate NX-AS-401 has potential as a strong antimicrobial adjuvant.

scholarly journals Subinhibitory Concentrations of Mupirocin Stimulate Staphylococcus aureus Biofilm Formation by Upregulating cidA

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Ye Jin ◽  
Yinjuan Guo ◽  
Qing Zhan ◽  
Yongpeng Shang ◽  
Di Qu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Multidrug Resistant ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Biofilm Development

ABSTRACT Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.

scholarly journals Tannic Acid Inhibits Staphylococcus aureus Surface Colonization in an IsaA-Dependent Manner

2012 ◽  
Vol 81 (2) ◽  
pp. 496-504 ◽  
Author(s):  
David E. Payne ◽  
Nicholas R. Martin ◽  
Katherine R. Parzych ◽  
Alex H. Rickard ◽  
Adam Underwood ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Tannic Acid ◽  
Biofilm Formation ◽  
Antibiofilm Activity ◽  
Host Immune System ◽  
Dependent Manner ◽  
Content Type ◽  
Surface Colonization ◽  
Biofilm Development ◽  
Insight Into

ABSTRACTStaphylococcus aureusis a human commensal and pathogen that is capable of forming biofilms on a variety of host tissues and implanted medical devices. Biofilm-associated infections resist antimicrobial chemotherapy and attack from the host immune system, making these infections particularly difficult to treat. In order to gain insight into environmental conditions that influenceS. aureusbiofilm development, we screened a library of small molecules for the ability to inhibitS. aureusbiofilm formation. This led to the finding that the polyphenolic compound tannic acid inhibitsS. aureusbiofilm formation in multiple biofilm models without inhibiting bacterial growth. We present evidence that tannic acid inhibitsS. aureusbiofilm formation via a mechanism dependent upon the putative transglycosylase IsaA. Tannic acid did not inhibit biofilm formation of anisaAmutant. Overexpression of wild-type IsaA inhibited biofilm formation, whereas overexpression of a catalytically dead IsaA had no effect. Tannin-containing drinks like tea have been found to reduce methicillin-resistantS. aureusnasal colonization. We found that black tea inhibitedS. aureusbiofilm development and that anisaAmutant resisted this inhibition. Antibiofilm activity was eliminated from tea when milk was added to precipitate the tannic acid. Finally, we developed a rodent model forS. aureusthroat colonization and found that tea consumption reducedS. aureusthroat colonization via anisaA-dependent mechanism. These findings provide insight into a molecular mechanism by which commonly consumed polyphenolic compounds, such as tannins, influenceS. aureussurface colonization.

scholarly journals An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

2015 ◽  
Vol 197 (24) ◽  
pp. 3779-3787 ◽  
Author(s):  
Vanina Dengler ◽  
Lucy Foulston ◽  
Alicia S. DeFrancesco ◽  
Richard Losick
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Matrix Proteins ◽  
Exogenous Dna ◽  
Content Type ◽  
Cytoplasmic Proteins ◽  
The Matrix ◽  
Biofilm Matrix

ABSTRACTStaphylococcus aureusis an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA, and polysaccharide. The exact compositions and roles of the different components are not fully understood. In this study, we investigated the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase and DNase treatments severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase-treated biofilm, clumping was partially restored, confirming the crucial role of eDNA in the interconnection of cells. On the basis of our results, we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH.IMPORTANCEExtracellular DNA (eDNA) is an important component of the biofilm matrix of diverse bacteria, but its role in biofilm formation is not well understood. Here we report that inStaphylococcus aureus, eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required to link cells together in the biofilm. These results confirm previous studies that showed that eDNA is an important component of theS. aureusbiofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix.

scholarly journals A Global Transcriptomic Analysis ofStaphylococcus aureusBiofilm Formation Across Diverse Clonal Lineages

2020 ◽  
Author(s):  
Brooke R. Tomlinson ◽  
Morgan E. Malof ◽  
Lindsey N. Shaw
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Transcriptional Regulation ◽  
Real Time ◽  
Biofilm Formation ◽  
Ribosomal Proteins ◽  
Multiple Time ◽  
List Type ◽  
Bacterial Persistence ◽  
Biofilm Development

AbstractA key characteristic ofS. aureusinfections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation - adhesion, proliferation, maturation, and dispersal - the tightly orchestrated molecular events behind each stage, and what drives variation betweenS. aureusstrains, has yet to be unraveled. Herein we measure biofilm progression and dispersal in real-time across the 5 majorS. aureusCDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple time points, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of PNAG production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53% of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages.ImportanceStaphylococcus aureusis a highly virulent, opportunistic pathogen and a leading cause of both nosocomial and community acquired infections. Biofilms are associated with persistent, chronic infections and the capacity to form a biofilm varies phenotypically amongstS. aureusclonal lineages. The molecular regulation of biofilm formation has been a popular area of study for this pathogen, however, a comprehensive mapping of the regulatory processes and factors driving strain-specific variation has yet to be elucidated. This study presents transcriptomic analyses of five diverse methicillin-resistantS. aureusisolates during various stages of biofilm formation, tracked in real time. The transcriptomic profiles of all five isolates were compared to identify both core and unique networks of regulation. Importantly, much of what we currently know about biofilms is based on mature, preformed biofilm populations, with little known of the transient regulation driving attachment, proliferation, maturation, and dissemination. We address this issue by investigating transcriptional regulation during the early, establishing phase of biofilm formation, and compare global transcriptional regulation both temporally and across multiple clonal lineages. This study provides a launching point towards understanding the highly orchestrated regulation driving each phase of biofilm development and may inform on future strategies to combat biofilm-mediated infections.Data SummaryRNA sequencing results have been deposited to the NCBI Gene Expression Omnibus; GEO submission GSE163153 (url –https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163153)Overview and comparisons of gene expression within biofilm and planktonic cell populations for the variousStaphylococcus aureusstrains are shown in Table S1, S2, S3. Validation and visual depiction of these data can be found as Figure S2 and S3 respectively (available in supplementary material).

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