Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability

Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability Laurie Destruel , View ORCID Profile Sandrine Dahyot , Laurent Coquet , Magalie Barreau , Stéphanie Legris , Marie Leoz , View ORCID Profile Maxime Grand , Xavier Argemi , Gilles Prevost , Nicolas Nalpas , Emmanuelle Dé , View ORCID Profile Sylvie Chevalier , Martine Pestel-Caron doi: https://doi.org/10.1101/2025.07.25.666850 Laurie Destruel a Univ Rouen Normandie, Université de Caen Normandie, INSERM, Normandie Univ, DYNAMICURE UMR 1311 , F76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sandrine Dahyot b Univ Rouen Normandie, Univ Caen Normandie, INSERM, Normandie Univ, DYNAMICURE UMR 1311, CHU Rouen, Department of Microbiology , F-76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandrine Dahyot For correspondence: sandrine.dahyot{at}chu-rouen.fr Laurent Coquet c Univ Rouen Normandie, INSA Rouen Normandie, CNRS, Normandie Univ, PBS UMR6270 , F-76000 Rouen, France d Univ Rouen Normandie, Inserm, CNRS, HeRacLeS US 51 UAR 2026, PISSARO , 76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Magalie Barreau e Univ Rouen Normandie, Univ Caen Normandie, Normandie Univ, CBSA, UR4312 , F-76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stéphanie Legris a Univ Rouen Normandie, Université de Caen Normandie, INSERM, Normandie Univ, DYNAMICURE UMR 1311 , F76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marie Leoz a Univ Rouen Normandie, Université de Caen Normandie, INSERM, Normandie Univ, DYNAMICURE UMR 1311 , F76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maxime Grand a Univ Rouen Normandie, Université de Caen Normandie, INSERM, Normandie Univ, DYNAMICURE UMR 1311 , F76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maxime Grand Xavier Argemi f University of Strasbourg, UR3073, CHRU Strasbourg, FMTS, Instituts de Bactériologie et de Parasitologie de la Faculté de Médecine , F-67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gilles Prevost f University of Strasbourg, UR3073, CHRU Strasbourg, FMTS, Instituts de Bactériologie et de Parasitologie de la Faculté de Médecine , F-67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicolas Nalpas c Univ Rouen Normandie, INSA Rouen Normandie, CNRS, Normandie Univ, PBS UMR6270 , F-76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Emmanuelle Dé c Univ Rouen Normandie, INSA Rouen Normandie, CNRS, Normandie Univ, PBS UMR6270 , F-76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sylvie Chevalier e Univ Rouen Normandie, Univ Caen Normandie, Normandie Univ, CBSA, UR4312 , F-76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sylvie Chevalier Martine Pestel-Caron b Univ Rouen Normandie, Univ Caen Normandie, INSERM, Normandie Univ, DYNAMICURE UMR 1311, CHU Rouen, Department of Microbiology , F-76000 Rouen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF ABSTRACT To understand the mechanisms involved in the evolutionary success of Staphyloccocus lugdunensis clones, we compared the biofilm-forming ability of representative strains of the seven clonal complexes (CCs) in rich and iron-restricted conditions, and characterized the extracellular matrix (ECM) of two highly biofilm-forming strains under each condition. Over 90% of the 49 S. lugdunensis strains produced biofilm in both conditions, with a level of production depending on the iron availability and clonal lineage. Two behaviors were observed: a significantly higher production in rich medium than in iron-restricted medium for CC1, CC2, and some CC3 strains, and the opposite phenomenon for CC6 ones. Analysis of the ECM of two representative strains using confocal microscopy showed that biofilm of the CC3 strain in rich medium contained similar amounts of proteins, eDNA and polysaccharides while that of CC6 strain was predominantly proteinaceous. Under iron-restricted conditions, biofilm structure and composition of both strains completely differed from those obtained in rich conditions. The proteomic analysis of their biofilm ECM by liquid chromatography coupled to tandem mass spectrometry identified 321 proteins common to both strains, mainly intracellular and in particular ribosomal. Of note, 202 proteins differed between the strains in terms of abundance, with a higher proportion of membrane proteins in the CC3 strain. This study performed on a large cohort of strains shows that S. lugdunensis biofilm-forming capacity is strongly associated with CC and iron availability. This analysis of biofilm-associated proteins in S. lugdunensis opens the way to propose new molecular targets for anti-biofilm strategies. IMPORTANCE The ability of S. lugdunensis to produce biofilm is considered as a critical virulence factor. As biofilm is strongly associated with persistence and difficult-to-treat infections, characterizing biofilm production and composition, particularly in iron-deficient environments encountered during infection, can provide a better understanding of therapeutic failures. Our work is the first to be carried out on such a large collection of S. lugdunensis clinical strains. It shows that this species is a strong biofilm producer, even in an iron-deficient environment, and that the composition of its matrix varies according to both genetic background of the strain and environmental conditions. Moreover, investigating the biofilms protein matrix of two S. lugdunensis strains provides insights into identification of potential targets for biofilm eradication. INTRODUCTION Staphylococcus lugdunensis is a particularly virulent species among coagulase-negative Staphylococci (CoNS), with a clinical pathogenic potential quite similar to that of Staphylococcus aureus ( 1 ). S. lugdunensis can cause a wide range of infections, including skin and soft tissue infections, bone and joint infections, catheter-related infections, and particularly aggressive and destructive infective endocarditis ( 2 ). To monitor the epidemiology of these infections, typing methods have been developed, such as the multilocus sequence typing (MLST), which defines sequence types (STs) grouped into clonal complexes (CCs) ( 3 , 4 ). Worldwide, seven CCs have been described, and interestingly, CC1 and CC3 were mainly identified in hospital settings, both in Asia and in France ( 4 – 7 ). To date, S. lugdunensis pathogenesis remains unclear, with few well-characterized virulence factors ( 1 , 2 , 8 ). Biofilm-forming ability appears as a characteristic of the species, and seems to be of particular importance in bone and joint infections (notably prosthetic joint infections) ( 9 , 10 ). Whole genome sequencing analyses have identified the presence of the ica locus in all the 21 S. lugdunensis genomes belonging to various CCs ( 11 ). The icaADBC locus encodes enzymes involved into biosynthesis of the poly-N-acetylglucosamine (PNAG) polysaccharide, which is commonly constitutive of the biofilm matrix of other CoNS and S. aureus strains ( 12 ). However, through biofilm dispersion assays, confocal laser scanning microscopy (CLSM) or immuno dot blot analyses, in vitro studies initially failed to detect the presence of PNAG in S. lugdunensis strains and revealed that biofilm matrix was mainly composed of proteins ( 13 , 14 ). Nevertheless, recent studies using the same methodological approaches have shown that polysaccharides along with proteins were the major components of S. lugdunensis mature 24h-biofilm ( 15 , 16 ). However, these studies were conducted on small collections of strains (11 to 38) not phylogenetically characterized, and biofilm analyses were carried out under laboratory optimal growth conditions in rich media ( 9 , 10 , 13 , 16 – 19 ). Only two studies have investigated biofilm formation under in vivo -like conditions, by analyzing biofilm-forming capacity of up to nine S. lugdunensis strains (clinical strains of unknown CC) in iron-deficient conditions ( 20 , 21 ). They revealed that the biofilm matrix comprised proteins and not PNAG under these low-iron growth conditions, and demonstrated the particular role of the iron acquisition system (Isd) proteins in promoting biofilm in this restricted environment. Recently, Cho et al. have characterized for the first time the extracellular matrix (ECM) proteome of a S. lugdunensis strain (NCCP 15630, Korea, CC unknown) in rich medium in comparison with planktonic condition, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) ( 22 ). Interestingly, among proteins identified only in biofilm were proteins involved in iron transport, like ferrous iron transport B and the probable heme-iron transport system permease protein IsdF, as well as a DUF5084 domain-containing protein ( 22 ). In this context, we first sought to further explore the role of iron-restricted conditions in biofilm production in a large collection of S. lugdunensis phylogenetically characterized strains. Second, we investigated the biofilm composition by CLSM as well as the ECM proteome by LC-MS/MS of two strong biofilm-producing strains belonging to different CCs. MATERIALS AND METHODS Bacterial strains Forty-nine S. lugdunensis strains were included in this study: 12 carriage strains and 37 responsible for human infections (Supplementary Table 1) ( 3 , 4 , 23 ). Forty-eight strains originated from diverse French University Hospitals (Bordeaux, Lyon, Montpellier, Nancy, Nantes, Rouen, Strasbourg, Tours) and one from Sweden (Kronoberg County). Strains were previously characterized by MLST ( 24 ) and/or fbl -typing ( 4 ). Forty-five strains belonged to 7 CCs (11 CC1, 5 CC2, 11 CC3, 4 CC4, 5 CC5, 5 CC6, and 4 CC7) and 4 were singleton STs (1 ST13 and 2 ST28) (Supplementary Table 1) . S. epidermidis ATCC 35984 (RP62A) strain was used as a biofilm producing control strain ( 25 ). Growth curve assays Growth kinetics of the 49 S. lugdunensis strains were performed in rich medium (trypticase soy broth, TSB, Bio-Rad, Marnes-la-Coquette, France) and in iron-depleted medium (RPMI 1640, Sigma-Aldrich, Saint-Louis, Missouri, USA) with 1% of casamino acids (Sigma-Aldrich) (RPMI). Overnight cultures were incubated in TSB or RPMI under shaking (150 rpm) at 37°C and then diluted to OD 600 of 0.01. Aliquots (200 µL per well) of standardized samples were inoculated in triplicate in a 96-well polystyrene microtiter plate. OD 600 measurement was performed every 15 min with the Spark® multimode microplate reader (Tecan, Männedorf, Switzerland) over 24h at 37°C under continuous double-orbital agitation (108 rpm). Data were obtained from biological triplicates. Generation time and maximal growth rate were determined with the R © software (v.3.6.0). Statistical analyses were performed by Kruskall-Wallis and post hoc Dunn’s test on GraphPad Prism © software. Biofilm formation Biofilm formation was assessed by a crystal violet staining assay (adapted from Stepanovic et al. 2007) ( 25 ) in TSB supplemented with 1% glucose (TSBG) and in RPMI for all the 49 S. lugdunensis strains and the control producing strain S. epidermidis ATCC 35984 ( 25 ). Overnight cultures were diluted to obtain an OD 600 of 0.01. Microtiter plates (96-wells) were filled in triplicate with 200 µL of bacterial suspension and incubated for 24h at 37°C under static conditions. Supernatants were removed and wells were washed once with sterile water. Remaining adherent cells were stained for 20 min with 200 µL of 0.5% crystal violet (Sigma-Aldrich) at room temperature, then washed twice with sterile water, and crystal violet was solubilized by ethanol 96% for 20 min. OD 590 was measured with the Spark® multimode microplate reader (Tecan). For each plate, three wells contained medium alone (blank). Four biological replicates were performed. Statistical analyses were performed by Wilcoxon Mann Whitney test on R © software. For each strain, the average OD 590 values for all strains were compared to the cut-off value (ODc). ODc was calculated using the following formula: ODc = average OD blank + 3 x standard deviation blank . Strains were classified as non-producer (OD 590 ≤ ODc), weak biofilm producer (ODc < OD 590 ≤ 2 x ODc), moderate producer (2 x ODc < OD 590 ≤ 4 x ODc) and strong producer (4 x ODc < OD 590 ) as previously described by Stepanovic et al. ( 25 ). Biofilm inhibition assays Assays were performed for a CC3 (03MC) and a CC6 (SL-55) strain using a method adapted from Panda and Singh study ( 26 ). The 24h-biofilm was grown in TSBG in 24-wells microplates. Solutions composed of proteinase K (100 µg/mL, Sigma-Aldrich), sodium metaperiodate (100 µg/mL, Sigma-Aldrich) or DNAse I (100 µg/mL, Sigma-Aldrich) were added onset of the culture to determine the amounts of proteins, exopolysaccharides and eDNA, respectively. Wells without enzymatic or chemical treatment were used as controls. Wells were then treated and stained with 0.5% crystal violet as described above. Data were obtained from three biological replicates. Results were expressed as ratio between mean of treated wells and mean of control wells. Statistical analyses were performed by Wilcoxon Mann Whitney test on R © software. Confocal Laser Scanning Microscopy (CSLM) A 48h-biofilm of the CC3 (03MC) and CC6 (SL-55) strains were grown in TSBG and RPMI in 24-well glass bottomed microplates (Greiner bio-one, Les Ulis, France). Briefly, the initial OD 600 was adjusted to 0.08. After 48h of incubation at 37°C, cells were washed with 300 µL of 0.9% NaCl to remove planktonic cells. The membrane integrity of the cells embedded in the biofilm was evaluated by LIVE/DEAD Bac Light Bacterial Viability Kit ® (Invitrogen, Waltham, Massachusetts, USA) according to the manufacturer’ instructions. Cells were stained by adding the green fluorescent probe Syto-9 (50 nM, Invitrogen). Biofilm matrix components were labeled using the red fluorescent SYPRO Ruby dye (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for proteins, the red fluorescent 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridinone (DDAO) (1 µM, Euromedex, Souffelweyersheim, France) for eDNA, and the blue fluorescent Calcofluor White (200 µg/mL, Sigma-Aldrich) for β1-3 and β1-4 exopolysaccharides. The CLSM observations of biofilms were performed using a Zeiss LSM710 confocal microscope (Carl Zeiss Microscopy, Oberkochen, Germany) using a x63 oil immersion objective. Images were taken every 0.5 µM. For visualization and processing of 3D images, the Zen 2.1 SP1 zen software (Carl Zeiss Microscopy) was used. Data were obtained from three biological replicates. Quantitative analysis of images was performed with COMSTAT software ( https://www.comstat.dk ) ( 27 , 28 ). ECM proteins characterization Matrix extraction ECM proteins of the CC3 (03MC) and CC6 (SL-55) strains were extracted according to an adaptation of the protocol of Chiba and coll. ( 29 ). Overnight cultures were diluted to obtain OD 600 of 0.01. Briefly, the biofilm was grown in 100 mL of TSBG for 24h in an Erlenmeyer flask containing 2g of glass wool under agitation (30 rpm) at 37°C. Glass wool was rinsed in 100 mL of sterile water. The ECM was extracted with 15 mL of 1.5 M NaCl. After gently shaking for 5 min on a roller shaker, bacterial cells were removed by centrifugation (5000 g for 15 min) and filtered through a 0.22 µM filter. Then, the ECM was precipitated overnight at 4°C in three volumes of 96% ethanol. Finally, the ECM was concentrated by centrifugation (9000 g for 30 min at 4°C), washed in 70% ethanol, air-dried and resuspended in water. The experiments were performed in biological quadruplicate for each strain. Trypsin digestion and nano LC-MS/MS ECM extracts (25 μg) were incubated overnight at 37°C with dispersin B (20 ng/µL) and loaded on SDS-PAGE gel (5% polyacrylamide) and migrated for 45 min at 20 mA. Proteins were stained with Coomassie blue. A single band containing all the extracted proteins was excised for endoprotease digestion carried out with an automated system (MultiPROBE II, PerkinElmer, Waltham, Massachusetts, USA). The protein bands were washed several times in water, dehydrated with acetonitrile, dried, incubated in a reductive solution (dithiothreitol 5 mM) and alkylated in iodoacetamide 25 mM. Then, the trypsin digestion was performed overnight with 1 µg of trypsin (Promega, Madison, Wisconsin, USA). The resulting peptides were recovered from the gel by incubation twice for 15 min in acetonitrile and once for 15 min in a 0.1% TFA (TriFluoroacetic Acid) solution. Peptides were dried, concentrated in 0.1% TFA solution and quantified by colorimetric peptide assay (Pierce Quantitative Peptide Assays, Thermo Fisher Scientific). For mass spectrometry analysis, 0.2 µg of protein digests were injected in an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) equipped with a nano-ESI source coupled to a nanoliquid chromatography (Easy-nLC II, Thermo Fisher Scientific). Peptides were separated by using a reversed phase C18 column (NikkyoTechnos, Japan) with a linear gradient of 15% to 55% of B (mobile phase A: water/0.1% TFA and phase B: Acetonitrile/0.1% TFA) over 120 min. The mass spectrometer was operated in data dependent mode to automatically switch between Orbitrap-MS (from m/z 300 to 2000) and LTQ-MS/MS acquisition. Protein identification and quantification Mass spectrometry data (raw data files) were processed using Progenesis QI software (Waters, Nonlinear Dynamics). Briefly, after peptide map alignment and normalization, the analysis of variance (ANOVA) with statistic filters was performed to select peptides showing significant and reproducible difference expression levels. When the p- value was < 0.05, the associated MS/MS spectra were exported for peptide identification with Mascot (Matrix Science v2.6.0) against the NCBI database restricted to S. lugdunensis HKU09-01 (RefSeq assembly: GCF_000025085.1). The identification searches were performed with variable modifications for oxidation of methionines, carbamidomethylation of cysteines, pyro-glutamate (Q and E) and with a maximum of 2 missed cleavages. MS/MS spectra were searched with a mass tolerance of 5 ppm for precursor ions and 0.35 Da for MS/MS fragments. Only peptides exhibiting significant Mascot individual ion score were retained. The proteins abundance was calculated by summing the abundances of all associated identified peptides. We first selected proteins identified from at least 2 peptides, and second compared the abundance of proteins using an ANOVA with a p- value 0.8 between the two strains. In addition, only the proteins showing a 1.8-fold ratio between the two strains was considered as significantly changing in abundance. Proteins’ cellular locations was predicted using the NCBI database and the PSORT website ( http://www.psort.org/psortb ). Participation in biological functional pathways was determined using the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg ). RESULTS Iron restriction affects S. lugdunensis biofilm formation according to the phylogenetic lineage Growth assays were performed on 49 S. lugdunensis strains in TSB and RPMI to evaluate the potential impact of phylogenetic lineage on growth in rich and iron-restricted conditions. Mean generation time and maximal growth rate in TSB were 37.3 min ± 4.1 and 1.1 ± 0.1, respectively, and 80.7 min ± 33.1 and 0.6 ± 0.2 in RPMI. The generation time was 2.2 times higher and the maximum growth rate was 1.9 times lower in iron-restricted medium compared to TSB. No growth difference was observed according to the strains or the CCs, whatever the medium. In TSB, the final stationary OD 600 was 1.2 ± 0.4 while it was lower in RPMI (0.5 ± 0.2). Although strain-dependent, the maximum OD 600 did not seem to depend on the CC of the strains. We then searched for a correlation between clonal lineages and ability to form biofilm and investigated whether experimental condition of iron limitation was correlated to a greater ability to produce biofilm. In TSBG, almost all strains (47/49, 95.9%) produced biofilm, among which 40.8% ( n = 20) were strong-, 32.7% ( n = 16) moderate- and 22.5% ( n = 11) weak-biofilm producers ( Figure 1A ). The two strains that did not produce biofilm belonged to CC5. CC2 strains produced significantly ( p <0.05) more biofilm than strains of all other CC ( Figure 2 ). Download figure Open in new tab Figure 1. Biofilm formation of 49 S. lugdunensis strains in rich (TSBG) (A), or iron deficient medium (RPMI) (B). Biofilm formation was evaluated by crystal violet staining from four independent biological experiments. Strains were classified as strong-(4 x ODc < OD), moderate-(2 x ODc < OD ≤ 4 x ODc), weak-(ODc < OD ≤ 2 x ODc) and non-biofilm producers (OD ≤ ODc) according to the final OD value. ODc = Average OD 590 blank + 3 ∗ Standard Deviation blank ( 25 ). Strains were classified by CC. The two strains marked with a star are those whose biofilm was further characterized. Download figure Open in new tab Figure 2. Biofilm formation comparison of 49 S. lugdunensis strains according to the CCs (OD 590 means) in rich-(TSBG, full bars) and iron-restricted (RPMI, dotted bars) media. Error bars represent the standard deviation within each CC. Significance was identified as p < 0.05 (*) or p < 0.01 (**) by Kruskal–Wallis test and post hoc Dunn test. Data are the result of four independent biological experiments. In iron-restricted conditions (RPMI), 45/49 strains (91.8%) produced biofilm. However, strains produced less biofilm than in TSBG (OD 590 max = 0.359 ± 0.062 in RPMI versus 0.897 ± 0.072 in TSBG). 26.5% ( n = 13) were strong-, 49% ( n = 24) moderate- and 16.3% ( n = 8) weak-biofilm producers ( Figure 1B ). Strains from CC6 formed significantly more biofilm than those from CC1 ( p = 0.016), CC2 ( p = 0.008), CC3 ( p = 0.004), CC4 ( p = 0.016) and CC5 ( p = 0.032) ( Figure 2 ). Whatever the medium, the biofilm production was not correlated to the clinical context (infection vs carriage) or the presence of medical devices ( Supplementary Table 1 ). However, when comparing biofilm formation in TSBG and RPMI, two CC-related behaviors were observed. On average, CC1 ( p = 0.013) and CC2 ( p = 0.0079) strains produced significantly more biofilm in rich medium than in iron-restricted conditions, while CC6 strains ( p = 0.0079) were significantly more productive in RPMI ( Figure 2 ). Of note, a heterogeneous behavior was observed among the CC3, with some strains being much higher producer ( e.g . 03MC) in rich conditions ( Figure 1 ). Biofilm composition and architecture differ between two S. lugdunensis strains of different CC Two S. lugdunensis strains were selected for further biofilm composition analysis, due to their different behaviors: one CC3 strain (03MC, isolated from skin and soft tissue infection, strong-biofilm producer in rich medium and weak-producer in iron-restricted conditions) and one CC6 strain (SL-55, isolated from skin and soft tissue infection, moderate-producer in rich medium and strong-producer in iron-restricted conditions). To investigate the role of eDNA, extracellular proteins, and polysaccharides in the first steps of biofilm formation, DNAse I, proteinase K and metaperiodate were used to alter each of these potential matrix constituents. Both strains showed a significantly ( p < 0.05) reduced biofilm formation in presence of DNAse I and proteinase K when added at the initial point of inoculation in rich medium (TSBG) ( Figure 3 ), the CC3 strain being more affected than the CC6 one. When using a pretreatment by metaperiodate, the CC3 strain biofilm formation was significantly reduced ( p = 0.0049), which was not the case for the CC6 strain. Download figure Open in new tab Figure 3. CC3 (03MC) and CC6 (SL-55) S. lugdunensis strains biofilm inhibition assays in presence of DNAse I (100µL/mL), proteinase K (100µL/mL) and metaperiodate (100µL/mL), from three independent biological assays. Error bars represent the standard deviation of stained biofilm. Significance was identified as p < 0.01 (**) by Wilcoxon Mann Whithney test. The architecture, cell viability and ECM composition of a 48-h biofilm was further investigated using CLSM for both strains. In rich medium, CC3 and CC6 strains were able to form a flat and homogeneous biofilm, with a maximal thickness of 22,06 ± 0.63 µm and 26.85 ± 0.47, and an average thickness for live cells of 18.66 ± 0.41 µm and 15.06 ± 0.49 µm, respectively ( Supplementary figure 1 ). Of note, CC3 strain biofilm was more compact than that of CC6 strain with roughness of 0.12 versus 0.32, respectively. Live/dead labeling showed that CC3 strain biofilm was mainly composed of viable cells, representing about 72.5% of the total cells, while the biofilm of CC6 strain was mainly composed of dead cells (61.6%) ( Figure 4 ). Noticeably, in iron-restricted medium, CC6 strain displayed a biofilm with increased biovolumes, maximal and average thicknesses compared to CC3 strain. In this condition, the ratio of dead cells increased in CC3 strain biofilm, reaching 54.8% of the total population, while the rate of dead cells in CC6 strain biofilm (47%) decreased compared to TSBG ( Figure 4 ). Download figure Open in new tab Figure 4. CC3 (03MC) and CC6 (SL-55) S. lugdunensis strains biofilm formation of in rich (TSBG) and iron restricted media (RPMI). 3D representations of biofilm assessed by cellular live/dead staining and observed by CLSM for CC3 (03MC) and CC6 (SL-55) strains in TSBG and RPMI (A). COMSTAT image analysis of biofilm composition of living (green) and dead (red) cells (B). Data are the results of the analysis of 10 views from three independent experiments. Looking at the ECM of these biofilms, in rich medium, CC3 strain was composed of relatively similar amounts of proteins, eDNA and polysaccharides with average thickness of 2.01 ± 0.18 µm, 3.77 ± 0.56 µm, and 1.49 ± 0.39 µm respectively, while the ECM of the CC6 strain was predominantly proteinaceous (average thickness of 4.98 ± 0.30 µm) ( Figure 5 , Supplementary figure 1 ). In iron-restricted, CC3 strain ECM was composed of proteins and polysaccharides in similar proportions (average thickness of 3.4 ± 0.2 µm and 2.27 ± 0.25 µm respectively) and eDNA in higher amounts (5.51 ± 0.4 µm), while CC6 strain contained a lower proportion of polysaccharides (1.01 ± 0.1 µm) than proteins (2.86 ± 0.3 µm) and eDNA (2.26 ± 0.13 µm). CC3 strain produced significantly larger amounts of ECM than in TSBG ( p <0.05), with a lower proportion (not significant) of proteins than in TSBG. CC6 strain produced slightly lower amounts of ECM in RPMI, with a propensity for a lower proportion of proteins ( Figure 5 , Supplementary figure 1 ). Download figure Open in new tab Figure 5. Extracellular matrix of S. lugdunensis biofilm. 3D representations of S. lugdunensis biofilm after labelling of live cells, proteins, eDNA, and polysaccharides by Syto9 (green), SYPRO ruby (red), DDAO (red) and Calcofluor White (blue) respectively in TSBG (A) and RPMI (B). COMSTAT image analysis of biofilm matrix composition (eDNA, proteins, and polysaccharides) relative to the total bacterial density (C), and relative to the total matrix (D). Data are the results of the analysis of 10 views from three independent experiments. In rich media, the matrix protein composition of the two strains differs in terms of abundance ECM proteomes of the 24-h biofilm of CC3 and CC6 strains in TSBG were obtained after analysis of the matrix extracts by LC-MS/MS. A total of 321 proteins was identified ( i.e. proteins identified from at least 2 peptides) ( Supplementary table 2 ); no protein was specific to either strain. These proteins were mainly identified as intracellular (71.7%), membrane proteins (13.7%), and a few ones were extracellular (4%) (10.6% were of unknown location). Ribosomal proteins accounted for 12.1% ( n = 39) of total proteins ( Table 1 ). Among membrane proteins, eight were ABC transporters, six were two-component systems and four were LPXTG-containing surface proteins (SLGD_00094, IsdA; SLGD_0090, IsdB; IsdC; and SLGD_00478, a triacylglycerol lipase). In addition, 12 transcriptional regulators were identified, including two from the LytR family (SLGD_01820 and SLGD_00719). Of the 20 most abundant proteins, 11 were common to both strains ( Table 2 ). These included a LTA synthase family protein (SLGD_02116, lipoteichoic acid synthase), metabolism proteins, a LysM peptidoglycan-binding domain-containing protein (SLGD_02450, N-acetylmuramoyl-L-alanine amidase) and an autolysin (SLGD_02076, N-acetylmuramoyl-L-alanine amidase). It is noteworthy that two proteins (SLGD_02212 and SLGD_00206) were highly abundant in both strains (abundance > 1,000,000). According to the NCBI database, SLGD_02212 corresponded to a SA0570 family protein, and SLGD_00206 to an immunodominant staphylococcal antigen IsaB family protein. View this table: View inline View popup Download powerpoint Table 1. Localization of proteins identified in the ECM matrix of S. lugdunensis CC3 and CC6 strains. View this table: View inline View popup Table 2. The 20 most abundant proteins among all identified proteins ( n = 321) in the biofilm matrix of the CC3 and the CC6 strains in TSBG (list in order of abundance for each strain). Proteins common to both strains are in bold. NA: not applicable Functional pathways of the 321 proteins were obtained via KEGG ( Figure 6 ). The main functional category was linked to metabolism (72% of proteins), followed by genetic information processing (22%) and environmental information processing (4%). The most represented pathway in the metabolism category was the carbohydrate metabolism ( n = 98 proteins); in this pathway, proteins were strongly involved in glycolysis/gluconeogenesis ( n = 22) and pyruvate metabolism ( n = 14), but more rarely in citrate cycle and tricarboxylic acid (TCA) cycle ( n = 9). Download figure Open in new tab Figure 6. Functional classification according to KEGG pathways of the 321 proteins identified in the TSBG biofilm matrix of S. lugdunensis CC3 (03MC) and CC6 (SL-55) strains (full bars), and upregulated in CC3 strain (hatched bars) and CC6 strain (dotted bars). The number of proteins of each channel is indicated above each histogram bar. Of the 321 proteins identified, 119 were considered to be non-varying between the two strains, as they had a p -value >0.05 and/or a power >0.8 and/or a fold change <1.8. Of the remaining proteins, 148 were significantly more abundant in the CC3 strain and 54 in the CC6 strain ( Supplementary table 2 ). In both strains, the significantly more abundant proteins were mainly intracellular (74.3% and 66.7% for CC3 and CC6 strains respectively). We found a higher proportion of membrane proteins among the more abundant proteins in the CC3 strain (16.9%) than in the CC6 strain (5.6%) ( Table 1 ); these included IsdB (SLGD_0090), three ABC transporters and two phosphotransferase systems ( Supplementary table 2 ). Moreover, LytR (SLGD_01820) was present among the twenty most abundant overexpressed proteins found in CC3 strain ( Supplementary table 3 ). The most represented pathway in both strains was metabolism (97.3% [144/148] and 61.1% [33/54] of proteins for CC3 and CC6 strains, respectively), and more specifically carbohydrate metabolism ( Figure 6 ). A higher percentage of proteins involved in processing genetic information was observed for CC3 strain compared to CC6 strain (26.4% vs 3.7%, respectively), with most proteins (35/39) being involved in translation. DISCUSSION Biofilm is strongly associated with persistence and difficult-to-treat infections ( 10 , 30 , 31 ). Concurrently, iron acquisition is critical for pathogenic bacteria to survive, colonize and invade host tissues. We therefore evaluated the impact of iron-restricted conditions (RPMI), compared to rich medium (TSBG), on bacterial growth and biofilm-forming capacity of a large selection of S. lugdunensis clinical strains belonging to the seven CCs defined by MLST to date. This study reveals that S. lugdunensis is a biofilm-producing species in both iron-rich and iron-poor environments, with production levels varying among strains. In TSBG, our results are consistent with those of previous studies using a microtiter plate assay, such as Frank and Patel ( 13 ), Qian et al . ( 15 ), Missineo et al. ( 21 ), and Hagstrand Aldman et al. ( 10 ) studies in which all strains were biofilm producers, but greatly higher than the findings of Pereira et al. (60.9% producers) ( 17 ). In iron-restricted medium, our strains formed on average significantly less biofilm than in rich medium, probably in connection with a reduced growth rate in RPMI. However, some strains, particularly CC6 strains, formed significantly more biofilm in this in vivo like condition. This result is in link with the findings of Missineo et al. where their nine studied strains (unknown CC) were biofilm producers (100%) in similar conditions (RPMI with 0.3% glucose and 2 mM glutamine) ( 21 ). In the same way, Aubourg et al. reported that iron limitation promoted biofilm formation of the strain S. lugdunensis N920143, but this was obtained in different conditions (brain-heart infusion broth with 2,2’-dipyridyl as iron chelator) and on a single strain ( 20 ). Moreover, this study shows for the first time that the biofilm formation of S. lugdunensis is associated with the phylogenetic lineage, with two CC-dependent behaviors (CC1 and CC2 strains producing significantly more biofilm in rich medium than in iron-restricted medium, while the opposite was observed for CC6 strains). It is noteworthy that the high-prevalent clones CC1 and CC3 were, on average, strong-biofilm producers, but exhibited strain-to-strain heterogeneity. Similarly, for S. aureus , a correlation between biofilm phenotype and clonal lineages defined by MLST and/or spa typing has been described ( 32 – 34 ). We further investigated the composition and the structure of S. lugdunensis biofilm by selecting two strains that best represented these types of behaviors: one strain belonging to the high-prevalent clone CC3 (03MC, strong-biofilm producer in rich medium) and one CC6 strain (SL-55, strong biofilm producer in RPMI). The biofilm formation and composition varied according to the strain and culture medium, as previously pointed out ( 15 , 16 ). Biofilm inhibition assays results indicated that in rich media, both proteins and eDNA play key role in the development and the structural integrity of S. lugdunensis biofilms, while the role of polysaccharides is strain-dependent. Indeed, the matrix of the CC6 strain was essentially proteinaceous without exopolysaccharides, which could be linked to a frameshift of icaA gene resulting in an early stop in IcaA (involved in synthesis of N-acetylglucosamine oligomers, 114 amino acids instead of 407) ( https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_008728735.1/ ). Data obtained by CLSM showed that the two strains of S. lugdunensis of different clonal lineage presented different biofilms in terms of architecture and cell viability, depending on their growth medium. Notably, in iron-restricted conditions, the biofilm of the CC3 strain contained more dead cells, which could be related to higher eDNA level, while that of the CC6 strain contained more living cells ( 35 , 36 ). Second, the ECM of both strains contained proportionally less proteins but more polysaccharides than in rich medium, suggesting that polysaccharides would play a more relevant role in the structural integrity of the biofilm in RPMI, contrary to what was shown by Missineo et al . ( 21 ). However, it should be noted that in our study exopolysaccharides were stained using Calcofluor White (as described by Grecka et al . [( 37 )]), which marks β-1,3 and β-1,4 polysaccharide bonds, whereas staphylococcal ECM has been described to date as mainly composed of PNAG which contains β-1-6 linkages ( 38 ). Thus, this suggests that polysaccharides other than PNAG could compose the biofilm matrix of S. lugdunensis strains ( 39 ). Further studies should consider completing these CLSM analyses by using a marker targeting the beta 1-6 bonds of PNAG, such as wheat germ agglutinin ( 13 ). Studying the protein composition of the matrix is of importance for understanding bacterial lifestyle and developing new therapeutic options against biofilm-associated infections. Only one study recently investigated the protein composition of the biofilm matrix of one S. lugdunensis strain (not phylogenetically characterized) ( 22 ). In the present study, biofilm formation screening of phylogenetically characterized strains enabled us to select two strains based on their CC (CC3 and CC6) as well as their biofilm formation ability. Analysis of the ECM proteome of these two representative strains has identified a total of 321 proteins, all common to both strains, which is low compared to the 1,125 identified by Cho et al . ( 22 ). This result may be related to differences in biofilm growth conditions (24-h biofilm in TSBG in our study versus 72-h biofilm in TSB without glucose in Cho et al ’s study) as well as the extraction method used (chemical in our study and mechanical in Cho et al .’s study) ( 22 ). The vast majority of the proteins identified in our study were cytoplasmic (71%), similar to studies that characterized biofilm ECM of S. aureus and S. epidermidis strains (60% to 86.2% of intracellular proteins) ( 40 – 42 ). These intracellular proteins may originate (i) from cell lysis within the biofilm, which may be autolysis-dependant (in line with the identification with one extracellular autolysin in the ECM of our strains) ( 43 ), (ii) from non-classical protein export by an yet unknown pathway or (iii) from mechanical cell lysis during ECM extraction. Of the intracellular proteins identified here, 12.4% were ribosomal proteins, whereas this rate reached 27% in the matrix of S. epidermidis in Martinez et al . study ( 41 ). The accumulation of cytoplasmic proteins could correspond to a “moonlighting” strategy of bacteria to stabilize the biofilm structure ( 40 , 44 ). Moreover, in our study membrane proteins accounted for 13% of the proteins identified in the matrix biofilm of S. lugdunensis . Among these proteins, eight were ABC transporters, and three were LPXTG-containing surface proteins, including three Isd proteins (IsdA, IsdB and IsdC). This result is in agreement with Cho et al .’s findings, who identified ABC transporters, one LPXTG-containing surface protein and the probable heme-iron transport system permease protein IsdF among the most abundant biofilm proteins, suggesting their role in the S. lugdunensis ECM composition ( 22 ). Identification of Isd proteins under rich conditions suggests that their expression is not limited to iron-limiting medium, unlike what was initially shown for IsdC ( 20 , 21 ). Of note, among the 20 most abundant matrix proteins identified in the biofilm of both strains, was a LysM peptidoglycan-binding domain-containing protein which, in S. aureus , mediates adherence to the extracellular matrix ( 45 ). Interestingly, an LTA synthase was also found, essential for the synthesis of teichoic acids, which is important for S. aureus or S. epidermidis initial adhesion to biotic surfaces ( 12 ). Two proteins were identified as highly abundant in the matrix of both strains, but were classified as hypothetical proteins. These proteins have been described in S. aureus : one (SLGD_02212) is a protease (Clp) ( 46 ), this type of enzyme being involved in biofilm dispersal phase ( 12 ), and the other (SLGD_00206) is an IsaB analog protein, an extracellular nucleic acid binding protein whose role in virulence is not established in S. aureus ( 46 ). KEGG analyzes revealed that the biofilm matrix of the two S. lugdunensis strains mostly contained proteins involved in metabolic pathways, particularly in carbohydrate metabolism, as previously described in S. lugdunensis and other staphylococci ( 22 , 41 , 47 , 48 ). In particular, we have identified a large number of proteins involved in glycolysis and gluconeogenesis for the formation of pyruvate, in line with the results obtained for S. epidermidis ( 41 ) or S. aureus biofilms ( 48 ). Similarly to S. epidermidis ( 41 ), few TCA cycle proteins were identified in our study in the ECM of S. lugdunensis biofilm, unlike S. aureus whose biofilm formation was initiated by metabolites produced by the TCA cycle ( 48 ). It would be interesting to compare proteomes of planktonic versus biofilm cultures of various strains of S. lugdunensis and to carry out metabolomic studies using metabolic techniques such as nuclear magnetic resonance ( 48 ) to confirm the involvement of these metabolic pathways biofilm formation in this species. Interestingly, ECM of the two S. lugdunensis strains differed in terms of proteins abundance. The higher number of proteins more abundant in CC3 strain compared to CC6 strain (148 vs . 54), including a higher number of membrane proteins, may be related to its strong ability to produce biofilm under rich conditions, as membrane proteins are involved in the early stages of biofilm formation ( 49 , 50 ). Genetic information processing proteins were more abundant in CC3 strain than in CC6 strain and included 22 ribosomal proteins, supporting the role of these proteins in biofilm formation. Furthermore, LytR, which was more abundant in CC3 strain biofilm, is a transcriptional regulator belonging to the two-component LytSR regulatory system, previously shown to be involved in biofilm formation in S. lugdunensis ( 51 , 52 ). In conclusion, our study is the first to investigate the biofilm-forming capacity of such a large collection of S. lugdunensis strains, representative of all the CCs, in rich- and iron-restricted media. We demonstrated for the first time that biofilm formation was strongly associated with CC affiliation and iron availability. Furthermore, the in-depth biofilm characterization of two strains belonging to two CCs using complementary approaches, including proteomic characterization of the ECM, confirmed notable differences in biofilm composition. 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Share Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability Laurie Destruel , Sandrine Dahyot , Laurent Coquet , Magalie Barreau , Stéphanie Legris , Marie Leoz , Maxime Grand , Xavier Argemi , Gilles Prevost , Nicolas Nalpas , Emmanuelle Dé , Sylvie Chevalier , Martine Pestel-Caron bioRxiv 2025.07.25.666850; doi: https://doi.org/10.1101/2025.07.25.666850 Share This Article: Copy Citation Tools Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability Laurie Destruel , Sandrine Dahyot , Laurent Coquet , Magalie Barreau , Stéphanie Legris , Marie Leoz , Maxime Grand , Xavier Argemi , Gilles Prevost , Nicolas Nalpas , Emmanuelle Dé , Sylvie Chevalier , Martine Pestel-Caron bioRxiv 2025.07.25.666850; doi: https://doi.org/10.1101/2025.07.25.666850 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41913) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13372) Ecology (19889) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40365) Molecular Biology (17163) Neuroscience (88540) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15130) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9818) Zoology (2269)