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

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

Variable number of tandem repeats in clinical strains of Haemophilus influenzae

An algorithm capable of identifying short repeat motifs was developed and used to screen the whole genome sequence available for Haemophilus influenzae, since some of these repeats have been shown to affect bacterial virulence. Various di- to hexanucleotide repeats were identified, confirming and extending previous findings on the existence of variable-number-of-tandem-repeat loci (VNTRs). Repeats with units of 7 or 8 nucleotides were not encountered. For all of the 3- to 6-nucleotide repeats in the H. influenzae chromosome, PCR tests capable of detecting allelic polymorphisms were designed. Fourteen of 18 of the potential VNTRs were indeed highly polymorphic when different strains were screened. Two of the potential VNTRs appeared to be short and homogeneous in length; another one may be specific for the H. influenzae Rd strain only. One of the primer sets generated fingerprint-type DNA banding patterns. The various repeat types differed with respect to intrinsic stability as well. It was noted for separate colonies derived from a single clinical specimen or strains passaged for several weeks on chocolate agar plates that the lengths of the VNTRs did not change. When several strains from different patients infected during an outbreak of lung disease were analyzed, increased but limited variation was encountered in al

Density and molecular epidemiology of Aspergillus in air and relationship to outbreaks of Aspergillus infection

After five patients were diagnosed with nosocomial invasive aspergillosis caused by Aspergillus fumigatus and A. flavus, a 14-month surveillance program for pathogenic and nonpathogenic fungal conidia in the air within and outside the University Hospital in Rotterdam (The Netherlands) was begun. A. fumigatus isolates obtained from the Department of Hematology were studied for genetic relatedness by randomly amplified polymorphic DNA (RAPD) analysis. This was repeated with A. fumigatus isolates contaminating culture media in the microbiology laboratory. The density of the conidia of nonpathogenic fungi in the outside air showed a seasonal variation: higher densities were measured during the summer, while lower densities were determined during the fall and winter. Hardly any variation was found in the numbers of Aspergillus conidia. We found decreasing numbers of conidia when comparing air from outside the hospital to that inside the hospital and when comparing open areas within the hospital to the closed department of hematology. The increase in the number of patients with invasive aspergillosis could not be explained by an increase in the number of Aspergillus conidia in the outside air. The short-term presence of A. flavus can only be explained by the presence of a point source, which was probably patient related. Genotyping A. fumigatus isolates from the department of hematology showed that clonally related isolates were persistently present for more than 1 year. Clinical isolates of A. fumigatus obtained during the outbreak period were different from these persistent clones. A. fumigatus isolates contaminating culture media were all genotypically identical, indicating a causative point source. Kn

Molecular typing of Salmonella typhi strains from Dhaka (Bangladesh) and development of DNA probes identifying plasmid-encoded multidrug-resistant isolates

Seventy-eight Salmonella typhi strains isolated in 1994 and 1995 from patients living in Dhaka, Bangladesh, were subjected to phage typing, ribotyping, IS200 fingerprinting, and PCR fingerprinting. The collection displayed a high degree of genetic homogeneity, because restricted numbers of phage types and DNA fingerprints were observed. A significant number of the S. typhi strains (67%) were demonstrated to be multiple drug resistant (MDR). The vast majority of the MDR strains were resistant to chloramphenicol, ampicillin, trimethoprim, streptomycin, sulfamethoxazole, and tetracycline (R type CATmSSuT), a resistance phenotype that has also frequently been observed in India. Only two strains displayed a distinct MDR phenotype, R type AT-mSSuT. Pulsed-field gel electrophoresis demonstrated the presence of large plasmids exclusively in the MDR strains of both R types. The plasmids present in the S. typhi strains of R type CATmSSuT could be conjugated to Escherichia coli and resulted in the complete transfer of the MDR phenotype. PCR fingerprinting allowed discrimination of MDR and susceptible strains. The DNA fragments enabling discrimination of MDR and susceptible S. typhi strains by PCR were useful genetic markers for identifying MDR encoded by large plasmids of the H1 incompatibility group