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Biomedical subjects

Holger Rohde

Publications and source records attributed to Holger Rohde.

At least 19 recordsLinked to original sources
PubMed2026

Detection of antibiotic heteroresistance in clinical microbiology: current and emerging methodologies.

BACKGROUND: Antibiotic heteroresistance (HR) is characterised by the coexistence of susceptible and resistant subpopulations within an apparently isogenic bacterial isolate. Because routine antimicrobial susceptibility testing (AST) primarily assesses the dominant population, HR may escape detection, potentially leading to discrepancies between laboratory susceptibility categorisation and the underlying bacterial population structure. OBJECTIVES: To provide a critical and practice-oriented evaluation of current and emerging methodologies for HR detection and to discuss their strengths, limitations, and potential for clinical implementation. SOURCES: Narrative review based on PubMed searches, complemented by screening of key reference lists and relevant EUCAST and CLSI documents. Peer-reviewed literature was prioritised. CONTENT: Phenotypic approaches, particularly population analysis profiling, remain the reference method for HR definition, but their labour-intensive workflows, long turnaround times, and limited standardisation restrict routine implementation. Alternative strategies, including modified AST assays, metabolic assays, and single-cell platforms, offer gains in speed or throughput but require broader validation. Molecular approaches such as quantitative PCR, droplet digital PCR, targeted deep sequencing, and whole-genome sequencing improve detection of minority resistance determinants. Emerging computational frameworks, including machine learning models integrating phenotypic and genomic data, represent a promising frontier for scalable HR prediction. IMPLICATIONS: Available evidence supports the clinical relevance of HR, although its association with adverse outcomes varies across bacterial species and antibiotic classes. Harmonised methodologies and clinically validated interpretive criteria are needed to support integration of HR assessment into routine diagnostics. Prospective multicentre studies and further standardisation, including engagement with EUCAST and CLSI, will be important to advance clinical implementation.

Antimicrobial resistance↗
PubMed2006

Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.

Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.

Adhesins, Bacterial↗
PubMed2006

Microbial interactions in Staphylococcus epidermidis biofilms.

Medical device-associated infections, most frequently caused by coagulase-negative staphylococci, especially Staphylococcus epidermidis, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is the most important factor in the pathogenesis of these infections, which regularly fail to respond to appropriate antimicrobial therapy. Progress in elucidating the factors functional in elaboration of S. epidermidis biofilms and the regulation of their expression with a special emphasis on the role of quorum sensing are reviewed. Significant progress has been made in recent years, which provides the rationale for developing better preventive, therapeutic and diagnostic measures.

Bacterial Physiological Phenomena↗
PubMed2005

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.

Adhesins, Bacterial↗
PubMed2005

Disintegration of Staphylococcus epidermidis biofilms under glucose-limiting conditions depends on the activity of the alternative sigma factor sigmaB.

To evaluate the role of the polysaccharide intercellular adhesin as an energy-storage molecule, we investigated the effect of nutrient limitation on S. epidermidis biofilms. The stability of established biofilms depends on sigma(B) activity; however, the slow decay of biofilms under conditions of nutrient limitation reveal its use as an energy-storage molecule to be unlikely.

Bacterial Proteins↗
PubMed2004

Recurrent Listeria monocytogenes aortic graft infection: confirmation of relapse by molecular subtyping.

Based on molecular typing methods, we identified a rare case of a recurrent L. monocytogenes infection resulting from an infected aortic prosthesis as detected by 18-F-Fluoro-d-deoxyglucose positron emission tomography (FDG PET). Our case highlights the usefulness of molecular typing and nuclear imaging methods for understanding L. monocytogenes pathogenesis and epidemiology.

Aortic Dissection↗
PubMed2004

Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses.

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.

Adaptation, Physiological↗
PubMed2004

RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR.

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 sigma(B) and biofilm formation, we generated different mutants of the sigma(B) 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 sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) 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 sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-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 sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

Bacterial Proteins↗
PubMed2004

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.

Acetylglucosamine↗
PubMed2004

Evaluation of the BD PHOENIX automated microbiology system for detection of methicillin resistance in coagulase-negative staphylococci.

The new BD PHOENIX automated microbiology system (Becton Dickinson Diagnostic Systems, Sparks, Md.) is designed for automated rapid antimicrobial susceptibility testing and identification of clinically relevant bacteria. In our study, the accuracy and speed of the BD PHOENIX oxacillin MIC determination for detecting methicillin resistance was evaluated for 200 clinical isolates of coagulase-negative staphylococci (CoNS). Compared to mecA PCR, the BD PHOENIX system detected methicillin resistance with a sensitivity of 99.2%. According to the actual NCCLS oxacillin MIC breakpoint of > or =0.5 microg/ml, the specificity was only 64.9%, attributable to false-positive results in 26 mecA-negative strains, including 16 non-Staphylococcus epidermidis strains. Alternative oxacillin breakpoints of > or =1, > or =2, and > or =4 microg/ml resulted in increased specificities of 83.8, 94.6, and 100% and high sensitivities of 99.2, 99.2, and 96.7%, respectively. Similarly, NCCLS broth microdilution oxacillin MICs exhibited a sensitivity of 100% but a low degree of specificity. However, the previous oxacillin MIC breakpoint of > or =4 microg/ml performed with a sensitivity of 98.4% and a specificity of 98.7%. BD PHOENIX oxacillin MIC results were available after 9 h for 40.5% of the examined CoNS strains and were completed after 17 h. Our results revealed the high reliability of the BD PHOENIX system as a phenotypic method for detection of resistance to oxacillin in mecA-positive CoNS. However, for the improvement of specificity, reevaluation of the optimal oxacillin MIC breakpoint for CoNS appears to be necessary.

Anti-Bacterial Agents↗
PubMed2004

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.

Adolescent↗
PubMed2003

Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants.

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.

Bacterial Adhesion↗
PubMed2003

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.

Adhesins, Bacterial↗
PubMed2002

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

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.

Agar↗