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Christof von Eiff

Publications and source records attributed to Christof von Eiff.

At least 19 recordsLinked to original sources
PubMed2007

A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins.

Bacterial intoxications represent a substantial public health concern with enterotoxins produced by Staphylococcus aureus among the most common causes of food poisoning. In addition to their role in the pathogenicity of food poisoning, staphylococcal enterotoxins have profound effects on the immune system as members of the family of pyrogenic toxin superantigens. As the classical diagnostic bioassays as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, there is a need for the timely identification of toxins by highly sensitive and specific methods. To combine the versatility of an enzyme immunoassay (EIA) with the amplification power of the PCR, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of staphylococcal enterotoxins A and B and compared to a commercially available EIA. A broadly applicable tool for signal amplification of pre-formed immunocomplexes was established by covalent binding of a reporter DNA to secondary detection antibodies. Therefore, the amino-modified reporter DNA was coupled successfully to N-succinimidyl-S-actyl-thioacetate-activated secondary detection antibodies. The qRT-iPCR was able to detect highly reproducibly as low as approximately 0.6 to 6 pg (4 to 40 amol/microl) of staphylococcal enterotoxin B and staphylococcal enterotoxin A, respectively. In conclusion, the qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for bacterial toxins, as demonstrated in this study for staphylococcal enterotoxins. The development of a stable antibody-DNA conjugate providing a universal signal amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for pre-formed antibody-antigen complexes.

Antibodies, Viral↗
PubMed2006

Reporter metabolite analysis of transcriptional profiles of a Staphylococcus aureus strain with normal phenotype and its isogenic hemB mutant displaying the small-colony-variant phenotype.

In this study, full-genome DNA microarrays based on the sequence of Staphylococcus aureus N315 were used to compare the transcriptome of a clinical S. aureus strain with a normal phenotype to that of its isogenic mutant with a stable small-colony-variant (SCV) phenotype (hemB::ermB). In addition to standard statistical analyses, systems biology advances were applied to identify reporter metabolites and to achieve a more detailed survey of genome-wide expression differences between the hemB mutant and its parental strain. Genes of enzymes involved in glycolytic and fermentative pathways were found to be up-regulated in the hemB mutant. Furthermore, our analyses allowed identification of additional differences between the normal-phenotype S. aureus and the SCV, most of which were related to metabolism. Profound differences were identified especially in purine biosynthesis as well as in arginine and proline metabolism. Of particular interest, a hypothetical gene of the Crp/Fnr family (SA2424) that is part of the arginine-deiminase (AD) pathway, whose homologue in Streptococcus suis is assumed to be involved in intracellular persistence, showed significantly increased transcription in the hemB mutant. The hemB mutant potentially uses the up-regulated AD pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Moreover, genes involved in capsular polysaccharide and cell wall synthesis were found to be significantly up-regulated in the hemB mutant and therefore potentially responsible for the changed cell morphology of SCVs. In conclusion, the identified differences may be responsible for the SCV phenotype and its association with chronic and persistent infections.

Arginine↗
PubMed2006

Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes.

Small colony variants (SCVs) of Staphylococcus aureus have been proposed to persist within vascular endothelium, thereby sustaining chronic infections. To identify the intracellular SCV location we infected primary human endothelial cells with various wild type and SCV strains of S. aureus and visualised maturation of phagosomes using live cell imaging. Staphylococci-containing phagosomes were matured by sequential and dynamic interactions with Rab5- and Rab7-positive vesicles. Within 45-60 min all internalised staphylococci accumulated in LAMP-1- and LysoTracker-enriched lysosomal organelles and remained there for up to 5 days. Recovery of most staphyloccocal strains was below 1% after a 24 h intracellular stay, indicating a high bactericidal activity of the endothelial cell lysosomes. However, the menadione auxotroph SCV strain JB1 displayed a recovery rate of 4% and, furthermore, through multiple intracellular passaging a subtype (JB1-P4) with a recovery rate of 25-30% could be generated. Interestingly, both JB1 and JB1-P4 also resided exclusively in lysosomes. Thus, on one hand we document effective bactericidal activity of human endothelial cell lysosomes towards staphylococci, and on the other hand we provide evidence that certain SCVs are capable to withstand this activity.

Endothelial Cells↗
PubMed2006

The small colony variant (SCV) concept -- the role of staphylococcal SCVs in persistent infections.

The recovery of small colony variants (SCVs) from clinical specimens was first described at the beginning of the last century. However, not until the past decade was an association of these variants with chronic, recurrent, and persistent infections such as chronic osteomyelitis and persistent skin and softtissue infection described. Since then, a number of reports and prospective studies have supported a pathogenic role for SCVs in disease. Biochemical and other characteristics of SCVs have suggested a link between electron-transport defective SCV strains and persistent infections, however, the strains examined were genetically undefined SCVs. Therefore, a stable mutant in electron transport was generated by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus aureus. This mutant showed characteristics typical of clinical SCVs such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Furthermore, the mutant was able to persist within cultured endothelial cells due to decreased a-toxin production. It was suggested that the intracellular location of this subpopulation might shield the variants from host defenses and antibiotics, thus providing one explanation for the difficulty in removing SCVs from host tissues. Therefore, a defect in the electron-transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly. Because of their fastidious growth characteristics, they are easily missed or misidentified in the clinical laboratory. Therefore, when an infection persists for weeks or months or fails to respond to antimicrobial therapy, clinicians as well as laboratory personnel should consider further efforts to search for this staphylococcal subpopulation.

Animals↗
PubMed2006

Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections.

Small colony variants constitute a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic traits. Phenotypically, small colony variants have a slow growth rate, atypical colony morphology and unusual biochemical characteristics, making them a challenge for clinical microbiologists to identify. Clinically, small colony variants are better able to persist in mammalian cells and are less susceptible to antibiotics than their wild-type counterparts, and can cause latent or recurrent infections on emergence from the protective environment of the host cell. This Review covers the phenotypic, genetic and clinical picture associated with small colony variants, with an emphasis on staphylococci, for which the greatest amount of information is available.

Aminoglycosides↗
PubMed2006

Biotyping of enterotoxigenic Staphylococcus aureus by enterotoxin gene cluster (egc) polymorphism and spa typing analyses.

Thirty-five Staphylococcus aureus strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the egc operon and spa typing. Nineteen spa types and seven different egc operons, including four putative new egc variants, were revealed. In 13 strains, allelic variants of sei and/or seg were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a sei variant, called the sei variant, was detected in six strains. In addition, the prototype sei was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described seg variant, also exhibiting several nucleotide exchanges. spa typing was more effective than REA egc grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same spa type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing egc-positive S. aureus strains.

Alleles↗
PubMed2006

Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model.

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.

Animals↗
PubMed2006

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype.

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.

Anti-Bacterial Agents↗
PubMed2006

Fourier-transform infrared spectroscopic analysis is a powerful tool for studying the dynamic changes in Staphylococcus aureus small-colony variants.

Infections due to small-colony variants (SCVs) of Staphylococcus aureus in patients with chronic and recurrent infections are an emerging problem; however, studies with this subpopulation are hampered by the fact that SCVs may exhibit unstable phenotypes, making them difficult to study, particularly in broth media. In this study, two S. aureus sets comprising the (i) normal and the (ii) SCV phenotype (clonal with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding site-directed mutants displaying the SCV phenotype (knockout of hemB) and (iv) their complemented mutants were examined by Fourier-transform infrared (FTIR) spectroscopy. Phenotypes were defined on solid and in broth media. Using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information resulted in a dendrogram with clear discrimination between SCV and normal phenotypes. The SCVs gave an FTIR fingerprint that was easily recognizable and that was much closer to other SCVs than to their parent strains. This technique offers for the first time a noninvasive approach to investigate dynamic processes of reversion of SCVs to the normal phenotype and vice versa. Thus, FTIR spectroscopy allowed a rapid and reproducible tool for the examination of different subpopulations of S. aureus on solid and in broth media for diagnostic and research purposes.

Bacterial Proteins↗
PubMed2006

Does nasal cocolonization by methicillin-resistant coagulase-negative staphylococci and methicillin-susceptible Staphylococcus aureus strains occur frequently enough to represent a risk of false-positive methicillin-resistant S. aureus determinations by molecular methods?

By analyzing the colonization of the anterior nares in cardiothoracic surgery patients on admission, nasal cocolonization by methicillin-susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci was detected in 8/235 (3.4%) specimens. Consequently, in a low-methicillin-resistant S. aureus (MRSA) setting, a molecular MRSA screening test targeting the mecA gene and an S. aureus-specific gene in parallel and applied directly to clinical specimens would be associated with an unacceptable positive predictive value of about 40%.

Bacterial Proteins↗
PubMed2006

Sequencing and staphylococci identification.

The emerging clinical importance of staphylococcal infections prompted us to establish a reference database for partial RNA polymerase B (rpoB; nucleotides 1444-1928) gene sequences from type strains of all staphylococcal species and subspecies. This database correctly identified 55 clinical staphylococcal isolates; all were correctly identified at the species level. At the subspecies level, rpoB misidentified only 2 isolates.

Animals↗
PubMed2005

Staphylococcus aureus small colony variants are resistant to the antimicrobial peptide lactoferricin B.

OBJECTIVES: To determine whether Staphylococcus aureus small colony variants (SCVs) are resistant to the antimicrobial peptide lactoferricin B. To assess if deficiency in transmembrane potential, a common characteristic of SCVs that are haemin- or menadione-auxotrophs, affects the uptake of the peptide into the bacterial cytoplasm. METHODS: A broth microdilution technique was used for susceptibility testing to determine the MIC of lactoferricin B for SCVs with three different auxotrophisms (haemin, menadione or thymidine) and their isogenic parent strains. Both clinical isolates and genetically defined mutants were used. The internalization of lactoferricin B in a hemB mutant and the respective parent strain was studied using transmission electron microscopy and immunogold labelling. RESULTS: All SCVs showed reduced susceptibility to lactoferricin B irrespective of their auxotrophy compared with their isogenic parent strains. The MIC for all SCVs was >256 mg/L, whereas the MICs for the parent strains ranged from 16-256 mg/L. Surprisingly, the hemB mutant contained significantly more lactoferricin B intracellularly than the respective parent strain. CONCLUSIONS: The resistance mechanism of SCVs towards the antimicrobial peptide lactoferricin B is presumably caused by the metabolic changes present in SCVs rather than by a changed transmembrane potential of SCVs or reduced uptake of the peptide.

Anti-Bacterial Agents↗
PubMed2005

Effectiveness of hydroxyapatite-vancomycin bone cement in the treatment of Staphylococcus aureus induced chronic osteomyelitis.

In the field of local application of antimicrobials, a number of novel drugs and/or new drug delivery systems have been developed in recent years. The present study aimed to investigate hydroxyapatite cement (HAC) as a carrier for vancomycin in the treatment of chronic osteomyelitis due to Staphylococcus aureus strains with various mechanisms of resistance. The release of vancomycin from standard test cylinders was determined in vitro and the efficacy of the delivery system was measured in vivo using a rabbit model of chronic osteomyelitis. First, powdered HAC was mixed with vancomycin at 80, 160 and 240 mg/g. After hardening, formed cylinders were eluted in phosphate buffer and antibiotic release was measured by agar diffusion. High levels of release (1512+/-318 to 1937+/-336 microg/ml) were obtained for 12 to 20 days depending on the dosage of vancomycin. Additionally, bone infection was induced in the tibia of 30 New Zealand white rabbits by injecting either a methicillin-resistant S. aureus strain (MRSA) or a S. aureus strain with a small colony variant (SCV) phenotype. After 3 weeks (chronic infection), all animals were treated by debridement. Moreover, group 1 (challenged with SCVs) and group 2 (challenged with MRSA) were treated by filling the marrow with HAC alone, whereas in groups 3 (SCVs) and 4 (MRSA) the marrow was filled with HAC/vancomycin (160 mg/g). After 6 weeks all animals were sacrificed. At 3 weeks, pathogens were detected in 24 of 30 animals. All swabs of the control groups, positive for S. aureus on day 21, were also positive on day 42 and S. aureus strains recovered were shown to be clonal to the strains used for induction of osteomyelitis. By contrast, no growth was found in the treatment group following 7 days of incubation in BHI bouillon. HAC/vancomycin-treated animals showed no histological evidence of infection on day 42. In the other groups, different stages of chronic osteomyelitis were found histologically. No local or systemic side effects due to HAC or vancomycin were seen. HAC is an effective carrier material for antibiotic compounds even in refractory infections due to MRSA or S. aureus SCVs.

Animals↗
PubMed2005

Thermonuclease gene as a target for specific identification of Staphylococcus intermedius isolates: use of a PCR-DNA enzyme immunoassay.

A PCR-DNA enzyme immunoassay (PCR-DEIA) was developed for identification of the coagulase-positive species Staphylococcus intermedius. Two PCR primers and a hybridization probe were designed to target specific sequences of the S. intermedius thermonuclease (nuc) gene. In addition to S. intermedius reference strains, the PCR-DEIA was tested using 295 veterinary and human S. intermedius isolates. A specific 933-bp DNA fragment was successfully amplified in 281 (94.9%) S. intermedius isolates. Five canine isolates showed an unexpected 2.8-kbp band. Except for 10 amplicons derived from equine, camel, and pigeon isolates, all positive PCR results (n = 288, 96.6%) were confirmed by the colorimetric microtiter plate DEIA hybridization. Isolates that failed both in amplification and DEIA hybridization were only observed in equine isolates (10/23, 43.5%). Except for the limitations with isolates of hoofed animals, the S. intermediusnuc PCR assay has potential for rapid identification of S. intermedius and differentiation from other coagulase-positive staphylococci including S. aureus.

Animals↗
PubMed2005

Antibacterial effects of extracorporeal shock waves.

Despite considerable knowledge about effects of extracorporeal shock-wave therapy (ESWT) on eukaryotic tissues, only little data are available concerning their effect on prokaryotic microorganisms. The objective of the present study was to determine the bactericidal activity as a function of energy flux density and shock-wave impulse number. Standardised suspensions of Staphylococcus aureus ATCC 25923 were exposed to different impulse numbers of shock waves with an energy flux density (ED) up to 0.96 mJ mm(-2) (2 Hz). Subsequently, viable bacteria were quantified by culture and compared with an untreated control. After applying 4000 impulses, a significant bactericidal effect was observed with a threshold ED of 0.59 mJ mm(-2) (p < 0.05). A threshold impulse number of more than 1000 impulses was necessary to reduce bacterial growth (p < 0.05). Further elevation of energy and impulse number exponentially increased bacterial killing. ESWT proved to exert significant antibacterial effect in an energy-dependent manner. Certain types of difficult-to-treat infections could offer new applications for ESWT.

Dose-Response Relationship, Radiation↗
PubMed2005

Comparative in vitro activity of ceftobiprole against staphylococci displaying normal and small-colony variant phenotypes.

The antistaphylococcal activity of ceftobiprole was compared with those of cefuroxime, linezolid, and moxifloxacin by using the agar dilution method. Apart from three strains with small-colony variant phenotypes, all Staphylococcus aureus isolates tested were inhibited by < or =2 microg/ml of ceftobiprole. This compound exhibited an excellent antistaphylococcal activity, comparable to that of linezolid.

Acetamides↗
PubMed2005

Comparable efficacies of the antimicrobial peptide human lactoferrin 1-11 and gentamicin in a chronic methicillin-resistant Staphylococcus aureus osteomyelitis model.

The therapeutic efficacy of an antimicrobial peptide, human lactoferrin 1-11 (hLF1-11), was investigated in a model of chronic methicillin-resistant Staphylococcus aureus (MRSA) (gentamicin susceptible) osteomyelitis in rabbits. We incorporated 50 mg hLF1-11/g or 50 mg gentamicin/g cement powder into a calcium phosphate bone cement (Ca-P) and injected it into the debrided tibial cavity, creating a local drug delivery system. The efficacy of hLF1-11 and gentamicin was compared to that of a sham-treated control (plain bone cement) (n=6) and no treatment (infected only) (n=5). The results were evaluated by microbiology, radiology, and histology. MRSA was recovered from all tibias in both control groups (n=11). On the other hand, hLF1-11 and gentamicin significantly reduced the bacterial load. Furthermore, no growth of bacteria was detected in five out of eight and six out of eight specimens of the hLF1-11- and gentamicin-treated groups, respectively. These results were confirmed by a significant reduction of the histological disease severity score by hLF1-11 and gentamicin compared to both control groups. The hLF1-11-treated group also had a significantly lower radiological score compared to the gentamicin-treated group. This study demonstrates the efficacy of hLF1-11 incorporated into Ca-P bone cement as a possible therapeutic strategy for the treatment of osteomyelitis, showing efficacy comparable to that of gentamicin. Therefore, the results of this study warrant further preclinical investigations into the possibilities of using hLF1-11 for the treatment of osteomyelitis.

Animals↗