Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono‐microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.     

Cloning of aas,a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties

A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L -alanine amidase and for an endo-β-N-acetyl-D -glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.     

Effect of allicin on the production of polysaccharide intercellular adhesin in Staphylococcus epidermidis

Aims: Polysaccharide intercellular adhesin (PIA) is the main agglutination agent in the biofilm forming strain Staphylococcus epidermidis. To find an explanation for the observed inhibition of biofilm formation by allicin, we studied the effect of allicin on PIA production in samples treated with sub MIC doses of allicin and compared this with a control culture without allicin. Methods and Results: Bacteria (Staph. epidermidis ATCC 35984) were grown in glass tubes, and PIA was extracted by vortex vibration using microbeads and NN dimethyl acetamide/LiCl as solvent. The extracts were filtered and passed through size exclusion columns. Chromatographic fractions were analysed with an excess of sodium metaperiodate and the excess was determined spectrophotometrically using 2,4,6‐tripyridyl‐s‐triazine. Conclusion: The amount of exopolysaccharides in samples previously treated with allicin is significantly lower than in the control. This finding suggests a specific enzymatic inhibition in PIA synthesis. Significance and Impact of the Study: This study provides an insight into the mechanism of biofilm formation, and is a biochemical model for PIA inhibition by allicin. The analysis proposed may be useful in studies of production of exopolysaccharides responsible for adherence and agglutination of Staph. epidermidis. Prevention of biofilm formation by allicin opens up a new field of in vitro studies and permits us to envisage future clinical applications.     

Biochemical properties of a novel metalloprotease from Staphylococcus hyicus subsp. hyicus involved in extracellular lipase processing.

Two extracellular proteases from Staphylococcus hyicus subsp. hyicus, ShpI and ShpII, have been characterized. ShpI is a neutral metalloprotease with broad substrate specificity; the gene has been cloned and sequenced. ShpII, characterized here, is mainly produced in the late logarithmic growth phase in contrast to ShpI, which is mainly produced in the late stationary growth phase. ShpII was purified from culture medium of S. hyicus by ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 34 kDa. The temperature optimum of ShpII was 55 degrees C, and the pH optimum was 7.4. ShpII, a neutral metalloprotease, was strongly inhibited by zinc and calcium chelators. The amino-terminal sequence of the active enzyme was similar to the corresponding region of a Staphylococcus epidermidis metalloprotease. The substrate specificity of ShpII was similar to that of thermolysin-like proteases, with the exception that ShpII also recognized aromatic amino acids. We demonstrated in vitro that ShpII, but not ShpI, cleaved the 86-kDa S. hyicus subsp. hyicus prolipase between Thr-245 and Val-246 to generate the mature 46-kDa lipase. Results of additional in vivo experiments supported the model that ShpII is necessary for the extracellular processing and maturation of S. hyicus subsp. hyicus lipase.     

Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device-related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA-negative strains are not uncommon, suggesting the existence of PIA-independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation-associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap-negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide-independent manner. In contrast, expression of full-length Aap did not lead to a biofilm-positive phenotype. Obviously, to gain adhesive function, full-length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by alpha(2)-macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein-dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.     

The biofilm-positive Staphylococcus epidermidis isolates in raw materials, foodstuffs and on contact surfaces in processing plants

Isolates from the “farm to fork” samples (182 isolates from 2779 samples) were examined genotypically (icaAB genes) and phenotypically (in vitro biofilm formation, typical growth on Congo red agar; CRA) with the aim to assess the risk of penetration of virulent strains of Staphylococcus epidermidis into the food chain. The contamination of meat and milk products was significantly higher in comparison with raw materials. Contamination of contact surfaces in the meat-processing plants was significantly lower than that of contact surfaces in the dairy plants. The ica genes (which precondition the biofilm formation) were concurrently detected in 20 isolates that also showed a typical growth on CRA. Two ica operon-negative isolates produced biofilm in vitro but perhaps by an ica-independent mechanism. The surfaces in the dairy plants and the milk products were more frequently contaminated with ica operon-positive strains (2.3 and 1.2 % samples) than the other sample types (0–0.6 % samples).     

Homologous and heterologous expression and maturation processing of extracellular glutamyl endopeptidase of Staphylococcus epidermidis

The extracellular serine endopeptidase GluSE (EC 3.4.21.19) is considered to be one of the virulence factors of Staphylococcus epidermidis. The present study investigated maturation processing of native GluSE and that heterologously expressed in Escherichia coli. In addition to the 28-kDa mature protease, small amounts of proenzymes with molecular masses of 32, 30, and 29 kDa were identified in the extracellular and cell wall-associated fractions. We defined the pre (M1-A27)- and pro (K28-S66)-segments, and found that processing at the E32-S33 and D48-I49 bonds was responsible for production of the 30- and 29-kDa intermediates, respectively. The full-length form of C-terminally His-tagged GluSE was purified as three proenzymes equivalent to the native ones. These molecules possessing an entire or a part of the pro-segment were proteolytically latent and converted to a mature 28-kDa form by thermolysin cleavage at the S66-V67 bond. Mutation of the essential amino acid S235 suggested auto-proteolytic production of the 30- and 29-kDa intermediates. Furthermore, an undecapeptide (I56-S66) of the truncated pro-segment not only functions as an inhibitor of the protease but also facilitates thermolysin processing. These findings could offer clues to the molecular mechanism involved in the regulation of proteolytic activity of pathogenic proteases secreted from S. epidermidis.     

Immunological detection of penicillin-binding protein 2'' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.     

The giant extracellular matrix-binding protein of Staphylococcus epidermidis mediates biofilm accumulation and attachment to fibronectin

Virulence of nosocomial pathogen Staphylococcus epidermidis is essentially related to formation of adherent biofilms, assembled by bacterial attachment to an artificial surface and subsequent production of a matrix that mediates interbacterial adhesion. Growing evidence supports the idea that proteins are functionally involved in S. epidermidis biofilm accumulation. We found that in S. epidermidis 1585v overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein (Embp) is necessary for biofilm formation. Embp is a giant fibronectin-binding protein harbouring 59 Found In Various Architectures (FIVAR) and 38 protein G-related albumin-binding (GA) domains. Studies using defined Embp-positive and -negative S.  epidermidis strains proved that Embp is sufficient and necessary for biofilm formation. Further data showed that the FIVAR domains of Embp mediate binding of S. epidermidis to solid-phase attached fibronectin, constituting the first step of biofilm formation on conditioned surfaces. The binding site in fibronectin was assigned to the fibronectin domain type III12. Embp-mediated biofilm formation also protected S. epidermidis from phagocytosis by macrophages. Thus, Embp is a multifunctional cell surface protein that mediates attachment to host extracellular matrix, biofilm accumulation and escape from phagocytosis, and therefore is well suited for promoting implant-associated infections.     

The evaluation of lipolytic activity of strains of Staphylococcus epidermidis and Staphylococcus haemolyticus

A total of 103 isolates of CNS (66 strains of S. epidermidis and 37 strains of S. haemolyticus) were investigated. Lipolytic activity of staphylococcal strains was determined by Tryptic Soy Agar containing Tween 20 or Tween 60. The 95.4% strains of staphylococci demonstrated the lipolytic activity on Tween 20 agar and the 89.4% of strains of staphylococci degradation ester of fatty acids on Tweens 60 agar. We detected that S. epidermidis strains (respectively 95,4%, 89,4%) produced lipases more frequently than S. haemolyticus strains (respectively 72,9%, 59,4%). Studies suggest that source of isolation from clinical materials (blood, wound and pus) does not have an influence on the ability hydrolysis esters.     

New categories designated as healthcare‐associated and community‐associated methicillin‐resistant Staphylococcus pseudintermedius in dogs

The incidence of MRSP has been increasing, and treatment options in veterinary medicine are limited. Few previous studies of MRSP have described the relationships between the genotypes, phenotypes, and clinical backgrounds of the isolates. To gain insight into the associations between the microbiological and clinical characteristics of MRSP, we analyzed 282 Staphylococcus pseudintermedius isolates from dogs. A total of 195 (69.1%) strains were identified as mecA‐positive MRSP and were classified into mainly two genotypes: SCCmec types III (II‐III) (52.8%) and V (37.4%). SCCmec type III MRSP strains were significantly correlated with hospital admission and antimicrobial therapy of the dogs, and exhibited a homogeneous genotype similar to sequence type 71‐MRSP, which is a globally endemic clone in dogs. In contrast, SCCmec type V MRSP strains were not highly correlated with hospital admission and antimicrobial therapy and exhibited genotypic and phenotypic heterogeneity. Properties of MRSP strains SCCmec types III and V were similar to those of HA‐ and CA‐MRSA, respectively. Therefore, we designated these isolates carrying SCCmec types III and V as HA‐MRSP and CA‐MRSP, respectively. Discrimination between HA‐ and CA‐MRSP by oxacillin MIC will provide useful information for treatment and infection control measures for canine MRSP infections.     

The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis.

The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to biofilm production of Staphylococcus epidermidis, which is thought to contribute to virulence in biomaterial-related infections. We purified a specific polysaccharide antigen of biofilm-producing S. epidermidis 1457 and RP62A, which was recently shown to have a function in the accumulative phase of biofilm production by mediating intercellular adhesion (D. Mack, M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs, Infect. Immun. 62:3244-3253, 1994). Following Sephadex G-200 gel filtration, this antigen was separated by Q-Sepharose chromatography into a major polysaccharide, polysaccharide I (> 80%), which did not bind to Q-Sepharose, and a minor polysaccharide, polysaccharide II (< 20%), which was moderately anionic. As shown by chemical analyses and nuclear magnetic resonance spectroscopy, polysaccharide I is a linear homoglycan of at least 130 beta-1,6-linked 2-deoxy-2-amino-D-glucopyranosyl residues. On average, 80 to 85% of them are N acetylated; the rest are non-N-acetylating and positively charged. Chain cleavage by deamination with HNO2 revealed a more or less random distribution of the non-N-acetylated glucosaminyl residues, with some prevalence of glucosaminyl-rich sequences. Cation-exchange chromatography separated molecular species whose content of non-N-acetylated glucosaminyl residues varied between 2 and 26%. Polysaccharide II is structurally related to polysaccharide I but has a lower content of non-N-acetylated D-glucosaminyl residues and contains phosphate and ester-linked succinate, rendering it anionic. Enzyme-linked immunosorbent assay inhibition with various monosaccharides revealed the beta-anomeric form and the acetylated amino group of the D-glucosaminyl residues as important for reactivity with the specific antiserum. The unbranched polysaccharide structure favors long-range contacts and interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular adhesion and biofilm accumulation. The structure of the polysaccharide is, so far, considered to be unique and, according to its function, is referred to as S. epidermidis polysaccharide intercellular adhesin (PIA).     

Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis

Abstract A gene ( fur ) for a Fur-like protein was identified on a 1.1 kb chromosomal DNA fragment of Staphylococcus epidermidis BN 280; the fur gene is followed by an open reading frame coding for the N-terminus of a putative Superoxide dismutase. Within the − 35 promoter region of both genes, a sequence motif was detected with low similarity to Fur-binding regulatory DNA segments, the so-called Fur boxes. Fur titration in Escherichia coli strain H1717 demonstrated that the E. coli Fur protein binds to the Fur box of the promoter region of the S. epidermidis fur gene. The S. epidermidis Fur protein was expressed in E. coli as indicated by the formation of inactive dimers with the chimeric repressor CI(N)-Fur(C) (Stojiljkovic I. and Hantke. K. (1995) Mol. Gen. Genet. 247, 199–205), but was not able to complement the Fur mutation in E. coli H1681.     

Impact of surface coating on the adherence of slime producing and nonproducing Staphylococcus epidermidis

The ability of Staphylococcus epidermidis to grow in the form of a biofilm not only facilitates its persistence in the host, but also allows it to survive at antibiotic concentrations several orders higher than the Minimum Inhibitory Concentration (MIC). We evaluated different surface treatments of hardened polystyrene in order to develop a model system for growth of S. epidermidis as a biofilm. We assayed for biofilm growth of S. epidermidis clinical isolates on unmodified polystyrene, on polystyrene modified by chemical abrasion and on polystyrene modified by sulfonation, using either Tryptic Soya Broth or Brain Heart Infusion as a growth medium. We concluded that sulfonated polystyrene and Brain Heart Infusion provided the best growth system for predicting the ability of a clinical isolate to form biofilm (Akaike value 23.680). Using this method, biofilm formation was detected in 14 (70%) of ica-positive strains and negative in 16 (80%) of ica-negative strains.     

The molecular characteristic of Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical sources

The aim of study was the molecular characteristic of S. aureus and S. epidermidis isolates obtained from skin surface, wounds, deep tissues of hospitalized patients and from skin surface of non-hospitalized patients. Genes encoding virulence factors were examined using PCR reaction and specific primers. Genes encoding adhesinsfnbA and cna and gene eta for epidermolytic toxin were mostly present in S. aureus isolates coming from wounds and deep tissues compared to these from skin surface. Gene atlE encoding autolysin of S. epidermidis was detected in all studied isolates, whereas gene icaAB was present in almost all isolates. Comparison of results obtained by PCR and conventional method of the resistance to methicillin estimation showed discrepances suggesting the need for using of both methods in some clinically difficult cases of S. aureus infection.