Johannes K.-M. Knobloch

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 α2‐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.

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.

Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms

Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.

Glucose-Related Dissociation between icaADBC Transcription and Biofilm Expression by Staphylococcus epidermidis: Evidence for an Additional Factor Required for Polysaccharide Intercellular Adhesin Synthesis

Biofilm formation in Staphylococcus epidermidis depends, in the majority of the strains, on the activity of the icaADBC locus. The expression of the operon that encodes the synthetic enzymes of the intercellular polysaccharide adhesin (PIA) depends on a variety of exogenic environmental conditions and is, at least in part, regulated by the alternative sigma factor sigma(B). We investigated the transcriptional regulation of the ica operon and the respective phenotypes expressed under growth conditions differing in the content of glucose in the growth medium. In the presence of glucose, S. epidermidis exhibited a PIA- and biofilm-positive phenotype whereas ica transcription was down-regulated in the postexponential and stationary phases of growth. Surprisingly, maximum transcription of ica was detectable in the stationary phase of growth in the absence of glucose despite the expression of a PIA- and biofilm-negative phenotype. In vitro enzymatic assays and phenotypic characterization showed that the abundant amount of ica mRNA was functionally active because induction of stationary-phase cells with glucose led to immediate PIA synthesis. Induction of biofilm formation could be completely inhibited by chloramphenicol, which, given at a later stage of biofilm accumulation, also inhibited further development of preformed biofilm, indicating that continuous translation of an additional, icaADBC-independent factor is required for the expression of a biofilm-positive phenotype.

Evaluation of different detection methods of biofilm formation in Staphylococcus aureus.

Abstract. The icaADBC gene locus of Staphylococcus aureus and its polysaccharide intercellular adhesin (PIA/PNSG) were recently identified, but biofilm formation has rarely been detected in vitro. In this study we evaluated a tissue culture plate (TCP) assay and a tube test, as well as Congo red agar, using the two basic media trypticase soy broth (TSB) and brain heart infusion (BHI) broth with different sugar supplements for detection of biofilm formation in 128 ica-positive S. aureus isolates. Of the S. aureus strains, 57.1% displayed a biofilm-positive phenotype under optimized conditions in the TCP test. The tube test correlated well with the TCP test for strongly biofilm-producing strains, whereas weak producers were not safely discriminated from biofilm-negative strains. Screening on Congo red agar displayed a strong correlation with the TCP and the tube test for only 3.8%, and is therefore not recommended for investigation of biofilm formation in S. aureus.

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

ABSTRACT The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis ofStaphylococcus 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 byS. 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.

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

ABSTRACT 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.

RsbU-Dependent Regulation of Staphylococcus epidermidis Biofilm Formation Is Mediated via the Alternative Sigma Factor σB by Repression of the Negative Regulator Gene icaR

ABSTRACT Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor σB and biofilm formation, we generated different mutants of the σB operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire σB operon, were deleted. Transcriptional analysis of sarA and the σB-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of σB activity, indicating regulation of σB activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via σB, indicating a crucial role for σB in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in σB or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking σB function but that icaA transcription is down-regulated in these mutants, indicating a σB-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a σB-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

Association Between Azithromycin Therapy and Duration of Bacterial Shedding Among Patients With Shiga Toxin–Producing Enteroaggregative Escherichia coli O104:H4

CONTEXT An outbreak of Shiga toxin-producing enteroaggregative Escherichia coli (STEC O104:H4) infection with a high incidence of hemolytic uremic syndrome (HUS) occurred in Germany in May 2011. Antibiotic treatment of STEC infection is discouraged because it might increase the risk of HUS development. However, antibiotic therapy is widely used to treat enteroaggregative E coli infection. In the German outbreak, a substantial number of patients received prophylactic azithromycin treatment as part of a therapeutic regimen with the C5 antibody eculizumab. OBJECTIVE To analyze the duration of bacterial shedding in patients with STEC infection who did and did not receive oral azithromycin therapy. DESIGN, SETTING, AND PATIENTS At a single center in Lübeck, Germany, 65 patients with STEC infection, including patients with HUS as well as STEC-infected outpatients without manifestation of HUS, were investigated between May 15 and July 26, 2011, and were monitored for a mean of 39.3 days after onset of clinical symptoms. MAIN OUTCOME MEASURE Carriage of STEC after azithromycin therapy. RESULTS Twenty-two patients received oral azithromycin and 43 patients did not receive antibiotic treatment. Among antibiotic-treated patients, long-term STEC carriage (>28 days) was observed in 1 of 22 patients (4.5%; 95% CI, 0%-13.3%) compared with 35 of 43 patients (81.4%; 95% CI, 69.8%-93.0%) who were not treated with antibiotics (P < .001). All 22 patients receiving azithromycin treatment had at least 3 STEC-negative stool specimens after the completion of treatment, and no recurrence of STEC was observed in these patients. As proof of principle, 15 patients who initially were not treated with antibiotics and were long-term STEC carriers were treated with oral azithromycin given for 3 days and subsequently had negative stool specimens. CONCLUSION Treatment with azithromycin was associated with a lower frequency of long-term STEC O104:H4 carriage.

[20] Genetic and biochemical analysis of Staphylococcus epidermidis biofilm accumulation

Publisher Summary This chapter discusses the genetic and biochemical analysis of Staphylococcus epidermidis accumulation. It is the most prominent coagulase-negative staphylococcal species that colonizes human skin and mucous membranes. It exhibits a low pathogenic potential in the normal human host. Coagulase-negative staphylococci ranks amongst the five most frequent causative organisms of nosocomial infections. The reason is a tremendous increase in the incidence of nosocomial sepsis and infections related to implanted foreign materials such as intravascular and peritoneal dialysis catheters, cerebrospinal fluid shunts, prosthetic heart valves and prosthetic joints, vascular grafts, cardiac pacemakers, and intraocular lenses. It is believed that the specific virulence of S. epidermidis in medical devicerelated infections is linked to an unusual ability to colonize polymer surfaces in multilayered biofilms. In vitro, a proportion of S. epidermidis strains produce a macroscopically visible, adherent biofilm on test tubes or tissue culture plates with morphology in scanning electron micrographs similar to that of infected intravascular catheters. Biofilm formation may be divided in two phases: primary attachment of staphylococcal cells to a polymer surface is a complex process involving various physicochemical factors; and the attached bacteria proliferate and accumulated in a multilayered biofilm.