Dietrich Mack

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis.

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA‐specific antiserum. PIA was shown to be a linear β‐1,6‐linked glucosaminoglycan composed of at least 130 2‐deoxy‐2‐amino‐D‐glucopyrano‐syl residues of which 80–85% are N‐acetylated, the rest being non‐N‐acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC‐carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co‐transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide‐polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N‐acetylglucosaminyltransferase involved in lipo‐chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N‐acetylglucosaminyltransferase activity in the formation

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.

Extended-spectrum β-lactamases: implications for the clinical microbiology laboratory, therapy, and infection control

Extended-spectrum β-lactamase (ESBL) producing gram-negative bacilli are a growing concern in human medicine today. When producing these enzymes, organisms (mostly K. pneumoniae and E. coli) become highly efficient at inactivating the newer third-generation cephaloporins (such as cefotaxime, ceftazidime, and ceftriaxone). In addition, ESBL-producing bacteria are frequently resistant to many classes of non-β-lactam antibiotics, resulting in difficult-to-treat infections. This review gives an introduction into the topic and is focused on various aspects of ESBLs; it covers the current epidemiology, the problems of ESBL detection and the clinical relevance of infections caused by ESBL-producing organisms. Therapeutic options and potential strategies for dealing with this growing problem are also discussed in this article.

Lactobacilli and bifidobacteria in the prevention of antibiotic-associated diarrhoea and Clostridium difficile diarrhoea in older inpatients (PLACIDE): a randomised double-blind, placebo-controlled, multicentre trial

BACKGROUND Antibiotic-associated diarrhoea (AAD) occurs most frequently in older (≥65 years) inpatients exposed to broad-spectrum antibiotics. When caused by Clostridium difficile, AAD can result in life-threatening illness. Although underlying disease mechanisms are not well understood, microbial preparations have been assessed in the prevention of AAD. However, studies have been mostly small single-centre trials with varying quality, providing insufficient data to reliably assess effectiveness. We aimed to do a pragmatic efficacy trial in older inpatients who would be representative of those admitted to National Health Service (NHS) and similar secondary care institutions and to recruit a sufficient number of patients to generate a definitive result. METHODS We did a multicentre, randomised, double-blind, placebo-controlled, pragmatic, efficacy trial of inpatients aged 65 years and older and exposed to one or more oral or parenteral antibiotics. A computer-generated randomisation scheme was used to allocate participants (in a 1:1 ratio) to receive either a multistrain preparation of lactobacilli and bifidobacteria, with a total of 6 × 10(10) organisms, one per day for 21 days, or an identical placebo. Patients, study staff, and specimen and data analysts were masked to assignment. The primary outcomes were occurrence of AAD within 8 weeks and C difficile diarrhoea (CDD) within 12 weeks of recruitment. Analysis was by modified intention-to-treat. This trial is registered, number ISRCTN70017204. FINDINGS Of 17,420 patients screened, 1493 were randomly assigned to the microbial preparation group and 1488 to the placebo group. 1470 and 1471, respectively, were included in the analyses of the primary endpoints. AAD (including CDD) occurred in 159 (10·8%) participants in the microbial preparation group and 153 (10·4%) participants in the placebo group (relative risk [RR] 1·04; 95% CI 0·84-1·28; p=0·71). CDD was an uncommon cause of AAD and occurred in 12 (0·8%) participants in the microbial preparation group and 17 (1·2%) participants in the placebo group (RR 0·71; 95% CI 0·34-1·47; p=0·35). 578 (19·7%) participants had one or more serious adverse event; the frequency of serious adverse events was much the same in the two study groups and none was attributed to participation in the trial. INTERPRETATION We identified no evidence that a multistrain preparation of lactobacilli and bifidobacteria was effective in prevention of AAD or CDD. An improved understanding of the pathophysiology of AAD is needed to guide future studies. FUNDING Health Technology Assessment programme; National Institute for Health Research, UK.

Structure, function and contribution of polysaccharide intercellular adhesin (PIA) to Staphylococcus epidermidis biofilm formation and pathogenesis of biomaterial-associated infections

Staphylococcus epidermidis is of major importance in infections associated with indwelling medical devices. The tight pathogenic association is essentially linked to the species ability to form adherent biofilms on artificial surfaces. Aiming at identifying novel targets for vaccination or therapy much effort has been made to unravel the molecular mechanisms leading to S. epidermidis biofilm formation. At present, polysaccharide intercellular adhesin (PIA) is the best studied factor involved in S. epidermidis biofilm accumulation. PIA is a glycan of beta-1,6-linked 2-acetamido-2-deoxy-D-glucopyranosyl residues of which 15 % are non-N-acetylated. PIA-producing S. epidermidis are widespread in clinical strain collections and PIA synthesis has been shown to be essential for S. epidermidis virulence. Moreover, PIA homologues have been identified in many other staphylococcal species, including the major human pathogen Staphylococcus aureus, and also Gram-negative human pathogens, suggesting that it might represent a more general pathogenicity principle in biofilm-related infections. In this review the current knowledge about the structure and biosynthesis of PIA is summarized. Additionally, information on its role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is discussed.

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.

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.

Staphylococcal Biofilm Exopolysaccharide Protects against Caenorhabditis elegans Immune Defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host–pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.