Polysaccharide Intercellular Adhesin Synthesis Research Articles

Biofilm Formation, icaADBC Transcription, and Polysaccharide Intercellular Adhesin Synthesis by Staphylococci in a Device-Related Infection Model

Biofilm formation of Staphylococcus epidermidis and S. aureus is mediated by the polysaccharide intercellular adhesin (PIA) encoded by the ica operon. We used a device-related animal model to investigate biofilm formation, PIA expression (immunofluorescence), and ica transcription (quantitative transcript analysis) throughout the course of infection by using two prototypic S. aureus strains and one S. epidermidis strain as well as corresponding ica mutants. During infection, the ica mutants were growth attenuated when inoculated in competition with the corresponding wild-type strains but not when grown singly. A typical biofilm was observed at the late course of infection. Only in S. aureus RN6390, not in S. aureus Newman, were PIA and ica-specific transcripts detectable after anaerobic growth in vitro. However, both S. aureus strains were PIA positive in vivo by day 8 of infection. ica transcription preceded PIA expression and biofilm formation in vivo. In S. epidermidis, both PIA and ica expression levels were elevated compared to those in the S. aureus strains in vitro as well as in vivo and were detectable throughout the course of infection. In conclusion, in S. aureus, PIA expression is dependent on the genetic background of the strain as well as on strong inducing conditions, such as those dominating in vivo. In S. epidermidis, PIA expression is elevated and less vulnerable to environmental conditions.

Detection of Virulence-Associated Genes Not Useful for Discriminating between Invasive and Commensal Staphylococcus epidermidis Strains from a Bone Marrow Transplant Unit

Because of their biofilm-forming capacity, invasive Staphylococcus epidermidis isolates, which cause the majority of nosocomial catheter-related bloodstream infections (BSIs), are thought to be selected at the time of catheter insertion from a population of less virulent commensal strains. This fact allows the prediction that invasive and contaminating strains can be differentiated via detection of virulence-associated genes. However, the hospital environment may pave the way for catheter-related infections by promoting a shift in the commensal bacterial population toward strains with enhanced virulence. The distribution of virulence-associated genes (icaADBC, aap, atlE, bhp, fbe, embp, mecA, IS256, and IS257), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated in S. epidermidis strains from independent episodes of catheter-related BSIs in individuals who have received bone marrow transplantation (BMT). The results were compared with those obtained for commensal S. epidermidis isolates from hospitalized patients after BMT and from healthy individuals, respectively. The clonal relationships of the strains were investigated by pulsed-field gel electrophoresis. icaADBC, mecA, and IS256 were significantly more prevalent in BSI isolates than in commensal isolates from healthy individuals. However, the prevalence of any of the genes in clonally independent, endogenous commensal strains from BMT patients did not differ from that in invasive BSI strains. icaADBC and methicillin resistance, factors important for the establishment of catheter-related infections, already ensure survival of the organisms in their physiological habitat in the hospital environment, resulting in a higher probability of contamination of indwelling medical devices with virulent S. epidermidis strains. The dynamics of S. epidermidis populations reveal that detection of icaADBC and mecA is not suitable for discriminating invasive from contaminating S. epidermidis strains.

Transcriptional activity of icaADBC is not correlated to the degree of biofilm formation in clinical ica-positive Staphylococcus epidermidis strains

Biofilm-formation in Staphylococcus epidermidis depends on the expression of the icaADBC operon encoding the enzymes required for the synthesis of polysaccharide intercellular adhesin (PIA). Different S. epidermidis strains vary widely in the degree of PIA and biofilm that they produce. In 11 clinical S. epidermidis strains we analyzed the biofilm-forming capacity in relation to the amount of ica expressed in static biofilm cultures. In mid-exponential growth phase no correlation could be detected between the level of ica transcription and the biofilm-forming phenotype. When the different strains were grown under conditions leading to a biofilm-negative phenotype, ica-expression was highly upregulated. Sequence analysis demonstrated that the observed differences were not due to major mutations in the ica promoter region but apparently to other strain-specific regulators.

Search for the insertion element IS256 within the ica locus of Staphylococcus epidermidis clinical isolates collected from biomaterial-associated infections

Staphylococcus epidermidis biofilm-forming strains produce a polysaccharide intercellular adhesin (PIA), which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants. PIA synthesis is regulated by the icaADBC locus. In vitro, by repeated subcultures of a biofilm-producing strain, the loss of the ability to produce biofilm appears associated with the insertion of the IS256 element into the ica locus. This study was aimed (i) to investigate if the five genes of ica locus are always all present in different strains of S. epidermidis, and (ii) to search if IS256 insertion naturally occurs in ica locus without making recourse to the experimental procedure of repeated subcultures of strains. 120 S. epidermidis clinical isolates from peri-prosthesis infections were investigated both by an original multiplex PCR analysis of the ica genes and by PCR amplification of the IS256 element. Also two reference strains (the biofilm-negative S. epidermidis ATCC 12228 and the biofilm-forming ATCC 35984 [RP62A]) and two biofilm-negative RP62A-derived acriflavin mutants (D9 and HAM892) were analysed. D9 e HAM892 were for the first time shown to contain in ica locus, at the base 3319, a 1300-bp insertion with a DNA sequence corresponding to IS256. Among the 120 clinical isolates, 51 (43%) turned out completely ica-positive, 69 completely ica-negative (57%). The genes of the ica locus appear, in all cases of the present collection, strictly linked each other, so they are either all present or all absent. In this collection, IS256 was present in eight out of the 69 ica-negative strains and in 34 out of the 51 ica-positive strains. IS256, also when present in bacterial genomic DNA, was never found inside the ica locus, thus suggesting that insertion/excision of this element is not a natural occurring mechanism for off/on switching of biofilm production.

Alcoholic ingredients in skin disinfectants increase biofilm expression of Staphylococcus epidermidis.

The pathogenesis of Staphylococcus epidermidis is correlated with biofilm formation. We investigated the effect of three common alcoholic skin disinfectants, ethanol, n-propanol and isopropanol, on the biofilm formation of 37 clinical, icaADBC-positive S. epidermidis isolates. In alcohol-supplemented media 18 strains displayed increased biofilm expression. Sixteen of 19 strains were generally incapable of biofilm formation. In three representative isolates, the increase in biofilm formation was paralleled by increased polysaccharide intercellular adhesin synthesis. Regarding the widespread use of alcoholic skin disinfectants, it is possible that the alcohol-inducible biofilm phenotype of S. epidermidis could add to the development of foreign body-related infections.

Differential Expression of Methicillin Resistance by Different Biofilm-Negative Staphylococcus epidermidis Transposon Mutant Classes

Biofilm formation mediated by polysaccharide intercellular adhesin (PIA) is the major virulence factor of Staphylococcus epidermidis and is often associated with methicillin resistance. Transposon Tn917 insertions leading to a biofilm-negative phenotype in the biofilm-producing S. epidermidis strain 1457 (mecA-negative) were transferred into the methicillin-resistant, biofilm-producing S. epidermidis 1057 (mecA-positive) by transduction. According to their phenotypes and genotypes, the mutants could be separated into genetic classes I to IV (D. Mack, H. Rohde, S. Dobinsky, J. Riedewald, M. Nedelmann, J. K. M. Knobloch, H.-A. Elsner, and H. H. Feucht, Infect. Immun. 68:3799-3807, 2000). All transductants of S. epidermidis 1057 had phenotypes for biofilm formation similar to those of the corresponding mutants of S. epidermidis 1457. With a mecA-specific probe, identical hybridization patterns were observed for wild-type S. epidermidis 1057 and all the transductants. There were minor changes in oxacillin MICs for Class II and III transductants compared to those for wild-type S. epidermidis 1057. On population analysis, S. epidermidis 1057 displayed a heterogeneous expression type of resistance with an oxacillin MIC of > or =6 microg/ml for more than 90% of the cells. An almost identical profile was observed with biofilm-negative class I mutants, where the transposon insertions inactivate the icaADBC gene locus essential for PIA synthesis. In contrast, class III mutants were more sensitive to oxacillin with a MIC of < or =1 microg/ml for more than 90% of the cells. The class IV mutant displayed homogeneous resistance with a MIC of > or =50 microg/ml for more than 90% of the cells. On oxacillin gradient plates, the class II mutant displayed decreased resistance. Apparently, different independent mutations leading to a biofilm-negative phenotype of S. epidermidis by influencing expression of icaADBC on the level of transcription significantly influence the expression of methicillin resistance. However, transcription of mecA was not significantly altered in the different transductants compared to the wild type, independent of mecA induction with oxacillin, indicating that other mechanisms influencing phenotypic expression of methicillin resistance are involved.

Correlation of biofilm expression types of Staphylococcus epidermidis with polysaccharide intercellular adhesin synthesis: evidence for involvement of icaADBC genotype-independent factors.

IcaADBC-encoded proteins mediate synthesis of the intercellular polysaccharide adhesin (PIA), which is essentially involved in Staphylococcus epidermidis biofilm formation. Seventy S. epidermidis isolates were investigated for their ability to form biofilm and synthesize PIA in different growth media including trypticase soy broth obtained from Becton Dickinson (TSBBBL), or Oxoid (TSB(OXOID)), and TSB(OXOID) supplemented with 0.5% N-acetylglucosamine, and for the presence of icaADBC. Dependent on the medium used (TSB(BBL) or TSB(OXOID)), the isolates exhibited a differential expression of PIA and biofilm formation, with 51 (72.85%) and 34 (48.57%) being biofilm positive, respectively. Using these growth media four different expression phenotypes were differentiated: similar quantities of biofilm formation in both TSBBBL and TSB(OXOID) (11 isolates, type A), significantly reduced biofilm expression in TSB(OXOID) compared to TSB(BBL) (23 isolates, type B), biofilm negative in TSB(OXOID) but biofilm producing in TSB(BBL) (17 isolates, type C) and biofilm negative in both media (19 isolates, type D). For all strains a biofilm-positive phenotype in a specific medium was closely linked to expression of PIA in that medium. All but one strain of expression type A-C and 7/19 expression type D strains were icaADBC positive. On the basis of restriction fragment length polymorphisms, the isolates were classified into two main icaADBC genotypes. There was no association between the observed biofilm-expression types and a defined icaADBC genotype. In the biofilm-negative S. epidermidis 5179, isolated from a ventriculo-atrial shunt infection, the insertion of IS257 interrupted the transcription of icaADBC, resulting in a PIA- and biofilm-negative phenotype. In all other icaADBC-positive, biofilm-negative isolates no major alterations of the icaADBC gene locus were identified. Obviously, expression of icaADBC, PIA synthesis and biofilm formation are integrated into a complex regulatory network involving other determinants independent of icaADBC genotype. Inactivation of icaADBC by IS elements is apparently a rare cause of a biofilm-negative phenotype in clinical S. epidermidis isolates.

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress.

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.

22] Efficient rna isolation method for analysis of transcription in sessile staphylococcus epidermidis biofilm cultures

Publisher Summary This chapter discusses the efficient RNA isolation method for the analysis of transcription in Sessile Staphylococcus epidermidis Biofilm Cultures. Staphylococcus epidermidis is a normal inhabitant of human skin and mucous membranes. With the increasing use of foreign biomaterials in medicine, these organisms have become one of the most frequently isolated pathogens in nosocomial infections. The specific pathogenicity of these skin commensals can be attributed to an unusual ability to colonize polymer surfaces in multilayered communities referred to as “biofilms.” The bacteria attach to a polymer surface, followed by accumulation of bacterial cells in multilayered cell aggregates encased in an amorphous glycocalyx. A polysaccharide intercellular adhesin (PIA), essential for bacterial accumulation mediates intercellular adhesion in these biofilms. Synthesis of PIA requires the expression of the icaADBC gene locus of S. epidermidis. Expression of biofilm formation and PIA by S. epidermidis depends significantly on different environmental factors, such as type of growth medium used, the presence of specific carbohydrates in the medium, and composition of the atmosphere. At least three independent regulatory gene loci control expression of the synthetic genes for PIA synthesis on the level of transcription. Transcriptional activity in biofilms of the S. epidermidis icaADBC locus and other gene loci relevant for biofilm formation under different physiologic growth conditions are unknown at present. Analysis of transcription under these conditions requires the recovery of extremely high-quality mRNA from established S. epidermidis biofilms.

Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation.

The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis of Staphylococcus epidermidis biomaterial-related infections. Using biofilm-producing S. epidermidis 1457 and transposon Tn917 carried on plasmid pTV1ts, we isolated nine isogenic biofilm-negative transposon mutants. Transduction by S. epidermidis phage 71 was used to prove the genetic linkage of transposon insertions and altered phenotypes. Mapping of the different transposon insertions by Southern hybridization and pulsed-field gel electrophoresis indicated that these were inserted in four unlinked genetic loci. According to their phenotypes, including quantitative differences in biofilm production in different growth media, in the amount of the polysaccharide intercellular adhesin (PIA) produced, in the hemagglutination titers, and in the altered colony morphology, the mutants could be separated into four phenotypic classes corresponding with the genetic classes. Synthesis of PIA was not detectable with class I and II mutants, whereas the amount of PIA produced reflected the residual degree of biofilm production of class III and IV mutants in different growth media. Chromosomal DNA flanking the transposon insertions of five class I mutants was cloned and sequenced, and the insertions were mapped to different locations of icaADBC, representing the synthetic genes for PIA. Expression of icaADBC from a xylose-dependent promoter in the different isogenic mutant classes reconstituted biofilm production in all mutants. In a Northern blot analysis no icaADBC-specific transcripts were observed in RNA isolated from mutants of classes II, III, and IV. Apparently, in addition to icaADBC, three other gene loci have a direct or indirect regulatory influence on expression of the synthetic genes for PIA on the level of transcription.

Induction of Staphylococcus epidermidis biofilm formation by environmental factors: the possible involvement of the alternative transcription factor sigB.

Staphylococcus epidermidis is a major cause of nosocomial infections associated with the use of indwelling medical devices1. Chronic infection of a prosthetic implant can serve as a septic focus that can lead to osteomyelitis, acute sepsis, and death, especially in immunocompromised patients 2 . The ability of S. epidermidis to generate thick biofilms on smooth polymer surfaces is regarded as the crucial step in the pathogenesis of these infections. It is associated with the production of a polysaccharide intercellular adhesin (PIA). The PIA represents a linear β -1,6-linked glucosaminoglycan and the bacteria become embedded in this slimy extracellular matrix during the accumulative phase of biofilm formation 3 . The enzymes involved in the PIA synthesis are encoded by the ica ADBC operon which was found to be widespread in S. epidermidis clinical isolates 4,5,6. The expression of this operon undergoes strong intrastrain variation and the investigation of the varying ica -expression revealed that this phenomenon is based on different genetic mechanisms including phase variation, ica -deletions and large chromosomal rearrangements 6,7. In this work we give evidence that S. epidermidis biofilm expression can also be

A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256.

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.