Application of PCR-mediated DNA typing in the molecular epidemiology of medically important microorganisms

This thesis describes the development, application and validation of the newer DNA analysis techniques within the field of microbiological epidemiology. Emphasis is placed on the use of the polymerase chain reaction (PCR), a test-tube technique enabling the amplification of (parts of) DNA molecules to enormous amounts. By comparing the genomes of microbes, insight in the mode of dissemination of given microorganisms can be obtained. Besides, this type of laboratory procedures also allows evolutionary studies, highlighting genomic variability perse. In the first chapter the teclmology will be introduced and the function of molecular typing within a microbiological laboratory will be explained. Subsequently, present day literature describing the application of PCR in microbial epidemiology will be summarised (chapter II). Chapters III and IV provide examples of the novel PCR approaches for molecular tracking of the protozoan parasites Naeg/eria spp. and Giardia duodenalis. The fact that the application of these techniques is not restricted to free-living amoebae and intestinal parasites is demonstrated in chapters V to VIII. Monitoring spread and persistence of fungal pathogens is performed, indicating the clinical relevance of genetic screening of a diversity of fungal agents, pathogens that went through a steep rise in clinical incidence during the past ten years. The last four chapters (IX to XII) describe the technical possibilities for and the clinical implications of molecular epidemiological studies on (methicillin-resistant) Staphylococcus aureus. Finally, chapter XIII integrates data presented in the previous chapters and gives a summary of current knowhow and sketches future developments

Comparison of four genotyping assays for epidemiological study of methicillin-resistant Staphylococcus aureus

Twenty-six methicillin-resistant Staphylococcus aureus strains were genetically differentiated by interrepeat PCR and the results compared with those of ribotyping, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA analysis obtained in a previous study for the same strains. The comparison showed that the PCR-mediated assays were as discriminatory as PFGE, whereas ribotyping was the least powerful genotyping method. Due to the ease of performance, PCR fingerprinting may become the method of choice for establishing clonal relationships among Staphylococcus aureus isolates

In vitro antibacterial and antibiofilm activities of chlorogenic acid against clinical isolates of stenotrophomonas maltophilia including the trimethoprim / sulfamethoxazole resistant strain

The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim / sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 g mL-1 and 16 to 32 g mL-1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia

Serotyping, ribotyping, PCR-mediated ribosomal 16S-23S spacer analysis and arbitrarily primed PCR for epidemiological studies on Legionella pneumophila

Fifty clinical and environmental isolates of Legionella pneumophila were typed serologically and by DNA fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR). Furthermore, variability in and around ribosomal operons was assessed by conventional ribotyping and PCR-mediated amplification of the spacer region separating the 16S and 23S genes. It appears that serotyping suffers from low resolution capabilities, and ribotyping and spacer PCR display intermediate resolving capabilities, whereas AP-PCR is more discriminating. Results from AP-PCR and both forms of ribotyping analysis correlate with epidemiological and environmental data. It is suggested that AP-PCR typing may be the method of choice for rapidly determining clonality among L. pneumophila isolates

Structure, toxicity and antibiotic activity of gramicidin S and derivatives

Development of new antibiotics is declining whereas antibiotic resistance is rising, heralding a post-antibiotic era. Antimicrobial peptides such as gramicidin S (GS), exclusively topically used due to its hemolytic side-effect, could still be interesting as therapeutic compounds. By modifying the amino-acid composition of GS, we synthesized GS analogues. We now show that derivative VK7 has a lower MIC (7.8–31.2 μg/ml, median 15.6 μg/ml) against strains of multi-drug resistant (MDR) Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa than GS has (3.9–62.5 μg/ml, median 31.3 μg/ml). Low MICs for both VK7 and GS were observed for Staphylococcus aureus and Enterococcus faecium

Mild Staphylococcus aureus skin infection improves the course of subsequent endogenous S. aureus bacteremia in mice

Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P) or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG) levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect