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P Gerner-Smidt

Publications and source records attributed to P Gerner-Smidt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Computerized analysis of restriction fragment length polymorphism patterns: comparative evaluation of two commercial software packages.

Two computerized restriction fragment length polymorphism pattern analysis systems, the BioImage system and the GelCompar system (Molecular Analyst Fingerprinting Plus in the United States), were compared. The two systems use different approaches to compare patterns from different gels. In GelCompar, a standard reference pattern in one gel is used to normalize subsequent gels containing lanes with the same reference pattern. In BioImage, the molecular sizes of the fragments are calculated from size standards present in each gel. The molecular size estimates obtained with the two systems for 12 restriction fragments of phage lambda were between 97 and 101% of their actual sizes, with a standard deviation of less than 1% of the average estimated size for most fragments. At the window sizes used for analysis, the GelCompar system performed somewhat better than BioImage in identifying visually identical patterns generated by electrophoretic separation of HhaI-restricted DNA of Listeria monocytogenes. Both systems require the user to make critical decisions in the analysis. It is very important to visually verify that the systems are finding all bands in each lane and that no artifacts are being detected; both systems allow manual editing. It is also important to verify results obtained in the pattern matching or clustering portions of the analysis.

Bacteriophage lambda↗
PubMed1997

[Extraintestinal Salmonella infections in four Danish counties. Inverse relationship between invasiveness and zoonotic serotypes].

The relationship between invasiveness and incidence of non-typhoid salmonellosis was ascertained retrospectively in a population of 1.9 million in four Danish counties during the three-year period 1992-1994. The study comprised 4175 cases diagnosed by culture either locally or at Statens Serum Institut, Copenhagen. Two hundred and forty-four patients had extraintestinal infections caused by 24 out of the total number of 101 different serotypes. Invasiveness ranged widely from 4% to > 90% for individual serotypes. Salmonella typhimurium and Salmonella enteritidis formed a high-incidence group (incidence rates > or = 25/100,000/year) compared to the remaining 22 serotypes (< 5/100,000/year). The low-incidence group was more invasive than the high-incidence group even when excluding Salmonella dublin, which is highly invasive, and including 212 cases of salmonelloses caused by serotypes isolated entirely from faeces (relative risk 1.54, 95% confidence limits 1.19 < RR < 2.00). The data corroborate a previous more limited study and indicate that previous exposure to salmonella and acquired immunity may play a role in the occurrence of extraintestinal non-typhoid salmonellosis.

Denmark↗
PubMed1997

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level.

The genus Acinetobacter is phenotypically rather homogeneous, but genotypically heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplified large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypically related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.

Acinetobacter↗
PubMed1997

Phylogenetic relationship of the twenty-one DNA groups of the genus Acinetobacter as revealed by 16S ribosomal DNA sequence analysis.

The inter- and intrageneric relationships of members of the genus Acinetobacter were investigated by performing a comparative sequence analysis of PCR-amplified 16S ribosomal DNAs (rDNAs) from 21 strains representing all of the DNA groups that have been described. Phylogenetic treeing confirmed that Acinetobacter spp. form a coherent cluster within the gamma subdivision of the class Proteobacteria that includes strains with overall levels of 16S rDNA sequence similarity of more than 94%. The analysis of intrageneric relationships suggested that the majority of the strains cluster in five clearly distinguishable clusters, and this conclusion was supported by the results obtained with the different methods used for phylogenetic analysis (i.e., the maximum-likelihood, parsimony, and distance matrix methods). The first cluster contains the representatives of DNA groups 2 (Acinetobacter baumannii) and TU13, whereas the second cluster comprises representatives of DNA groups 3, "Close To TU13," and "between 1 and 3." The representatives of closely related Acinetobacter DNA groups 8 (Acinetobacter twoffii) and 9 belong to the third cluster, which includes the representative of DNA group 6 as well. The fourth cluster is formed by DNA groups BJ15, BJ16, and BJ17, and the fifth cluster comprises DNA groups 1 (Acinetobacter calcoaceticus), BJ14, 10, and 11. Within the fifth cluster the 16S rDNA sequences of DNA group 10 and 11 strains are nearly identical. The representatives of DNA groups 4 (Acinetobacter haemolyticus), 5 (Acinetobacter junii), 7 (Acinetobacter johnsonii), 12 (Acinetobacter radioresistens), TU14, and TU15 form individual branches that are not significantly affiliated with any of the five clusters identified. Apart from the clustering of the most closely related DNA groups, the general topology of the distance dendrogram revealed some discrepancy with previous DNA-DNA hybridization data, which may point to the inadequacy of comparative 16S rDNA sequence analysis for reflecting true evolutionary relationships of closely related bacterial taxa. Important, however, was the presence of unique sequence motifs in each of the 21 different DNA groups studied, which may be useful for rapid differentiation of DNA groups of the genus Acinetobacter.

Acinetobacter↗
PubMed1997

Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods.

At least 19 genomic species are recognized as constituting the genus Acinetobacter. However, little is known about the natural reservoirs of the various members of the genus. An epidemiological study was therefore performed to investigate the colonization with Acinetobacter spp. of the skin and mucous membranes of 40 patients hospitalized in a cardiology ward and 40 healthy controls. Single samples were obtained once from each of nine different body sites, i.e., forehead, ear, nose, throat, axilla, hand, groin, perineum, and toe web. Identification of Acinetobacter isolates was achieved by using phenotypic properties and was compared to identification by amplified ribosomal DNA restriction analysis. Selected isolates were further investigated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ribotyping, and DNA-DNA hybridization. Plasmid profile analysis was used for epidemiological typing. Thirty patients (75%) and 17 controls (42.5%) were found to be colonized with Acinetobacter spp., and the colonization rates of patients increased during their hospital stay. The most frequently isolated species were Acinetobacter lwoffii (47%), A. johnsonii (21%), A. radioresistens (12%), and DNA group 3 (11%). In contrast, A. baumannii and DNA group 13TU, the most important nosocomial Acinetobacter spp., were found only rarely on human skin (0.5 and 1%, respectively) and their natural habitat remains to be defined. A good correlation between phenotypic and genotypic methods for identification of Acinetobacter spp. was observed, and only two isolates could not be assigned to any of the known DNA groups.

Acinetobacter↗
PubMed1997

Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp.

Seven laboratories in six European countries examined 40 isolates belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex to investigate whether standardized protocols and quality-controlled reagents could produce reliable, discriminatory, and reproducible PCR-based fingerprinting results. Four PCR protocols with different primers (primers DAF4, ERIC-2, M13, and REP1 + REP2) were used. The epidemiological conclusions reached by the participating laboratories were substantially correct, with 96.4% of the total isolate grouping allocations agreeing with the consensus view. All laboratories identified the main epidemiological clusters, and each laboratory also identified two non-outbreak-related isolates. There were no significant differences between the isolate grouping results obtained by the different protocols and with the different primers. Visual comparison indicated that the standardized protocols and reagents yielded reproducible fingerprint patterns, but with some variations in particular band intensities. Minor variations in fingerprint profiles were detected, but computer-assisted analysis of PCR fingerprints obtained on agarose gels demonstrated that 88.3 to 91.6% (depending on the source of DNA) of the patterns clustered correctly, while 96.4 to 98.9% of the patterns clustered correctly following automated high-resolution laser fluorescence analysis. Correlation of the patterns for isogenic isolates ranged from 83.3 to 86.6% but was slightly better (mean correlation, 87.1%) for centrally prepared DNA extracts than for DNA extracts prepared by individual laboratories (mean correlation, 84.7%). It was concluded that independently produced PCR fingerprint patterns can be obtained reproducibly for Acinetobacter spp. at the practical level if (i) quality-controlled reagents, (ii) standardized extraction of DNA, and (iii) standardized amplification conditions are used.

Acinetobacter↗
PubMed1997

Epidemiological study of Acinetobacter species isolated from an intensive care unit.

An epidemiological survey of the increase in Acinetobacter species isolates occurring in the intensive care unit of a Spanish teaching hospital during 1993 and 1994 was carried out. Different laboratory methods were used to find out, whether there was a genetic linkage. The isolates were divided into three main groups according to the resistance patterns to 11 drugs. Using API 20NE biotyping, eight different types were found. The two most common contained 20 and 11 isolates, respectively. Five different plasmid profile types were observed, although plasmids were only demonstrated in 40% of the isolates. Ribotyping with EcoRI, SalI and ClaI enzymes revealed 10, 9, and 8 different patterns, respectively. In total, 15 different ribotypes were identified using these three enzymes. Twenty-one isolates belonged to exactly the same ribotype, and 13 were associated with two highly related ribotypes. In the first ribotype, only five isolates harboured plasmids. The ribotyping method produced 100% typability and ribotypes were easy to compare; it also had taxonomic value. Ribotyping allowed us to determine the genetic linkage between Acinetobacter isolates recovered from ICU patients.

Acinetobacter↗
PubMed1996

WHO-sponsored international collaborative study to evaluate methods for subtyping Listeria monocytogenes: restriction fragment length polymorphism (RFLP) analysis using ribotyping and Southern hybridization with two probes derived from L. monocytogenes chromosome.

Seven laboratories participated in a WHO-sponsored international collaborative study, to evaluate methods for subtyping Listeria monocytogenes, by performing restriction fragment length polymorph sm (RFLP) analysis-based subtyping of an international study set of 80 strains of L. monocytogenes that included 22 epidemiologically related groups. The RFLP analysis was done by Southern hybridization with one of two types of probes found in multiple copies on the chromosome of L. monocytogenes. Six laboratories performed ribotyping. These laboratories used EcoRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or DNA as the probe for Southern hybridizations. The seventh laboratory used Ncil to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calculation of Simpson's index of diversity, ranged from 0.83 to 0.88 for the six laboratories. The discriminating power of the combination of two probes used by Laboratory 7 was 0.91. Ribotyping and the cloned probes used by Laboratory 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains (identified by other subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of serotypes 4b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from 80.0 to 100%. as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants.

Bacterial Typing Techniques↗
PubMed1996

WHO study on subtyping Listeria monocytogenes: results of phage-typing.

A multi-centered study on phage typing of Listeria monocytogenes was carried out using 80 cultures sent under code and tested in six different laboratories. Phage typing was performed using an international phage set in five laboratories and phage sets unique to two laboratories. Testing of cultures sent in duplicate showed similar levels of reproducibility to that previously reported. Analysis of results from groups of epidemiologically related cultures showed a high level of agreement in all laboratories. Patterns of phage susceptibility were relatively stable on retesting strains in the same laboratory after long periods of time. However, there was limited comparability between results obtained from testing the same cultures using the same phages in different laboratories. It is recommended that the phages in the international set be reviewed, and that better inter-laboratory reproducibility may be achieved by standardisation of phage suspensions, propagation strains and methodology, together with the use of centrally propagated phages.

Bacteriophage Typing↗
PubMed1996

High-frequency endonuclease (REA) typing: results from the WHO collaborative study group on subtyping of Listeria monocytogenes.

Eighty isolates of Listeria monocytogenes were typed by high-frequency restriction endonuclease analysis. Two laboratories participated in the study with two restriction enzymes each. The enzymes used were EcoRI, Hae1II, HhaI. and its isoschizomer Cfo1. EcoRI was less discriminatory than the other enzymes. The profiles generated by Hha1 and CfoI were not fully stable for some closely related isolates. The size and the number of restriction bands generated by Hha1 in one laboratory and its isoschizomer Cfo1 in another laboratory were directly comparable. This indicates that REA-typing may be used as a definitive typing method for Listeria monocytogenes if the typing procedure is standardized. The stability of the REA-types needs further elucidation in order to establish firm differentiation criteria for comparison of isolates.

Bacterial Typing Techniques↗
PubMed1996

Acinetobacter species identification by using tRNA spacer fingerprinting.

Identification of Acinetobacter spp. to the DNA group level by phenotypic techniques is problematic, and there is a need for an alternative identification method for routine use. The present study validated the suitability of a rapid identification technique based on tRNA spacer (tDNA) fingerprinting in comparison with that of a commercially available assay involving carbon source utilization tests (Biolog MicroStation System) for identifying the 21 DNA-DNA hybridization groups belonging to the genus. For this purpose, 128 strains identified previously by DNA-DNA hybridization were analyzed by both techniques. tDNA fingerprinting was highly reproducible and classified all strains into 17 groups. Six DNA groups belonging to the A. calcoaceticus-A. baumannii complex were grouped into two distinct clusters, indicating the high degree of genetic similarity within this complex. Strains of the more recently described DNA groups BJ13 to BJ16 were ambiguously grouped and displayed three pattern types. The software used with the commercial carbon source utilization method grouped the 128 strains into 12 clusters, explaining the less discriminatory power of this system. We conclude that tDNA fingerprinting offers a quick and reliable method for the routine differentiation of most Acinetobacter spp. at the subgenus level.

Acinetobacter↗
PubMed1996

Amplification and restriction endonuclease digestion of a large fragment of genes coding for rRNA as a rapid method for discrimination of closely related pathogenic bacteria.

By use of primers specific to conserved regions of the rRNA gene cluster, a discrete amplicon of approximately 5 kb was amplified by PCR from all 21 bacterial genera investigated. Subsequent endonuclease digestion of the PCR product with HaeIII distinguished between the three species of the human pathogen Francisella spp. on the one hand and four clinically relevant genomic groups of Acinetobacter spp. on the other hand. The described technique has the potential as a rapid method for discriminating between closely related species that are of clinical importance.

Acinetobacter↗
PubMed1996

Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak.

Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique profile. Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex.

Acinetobacter↗
PubMed1996

Comparison of outbreak and nonoutbreak Acinetobacter baumannii strains by genotypic and phenotypic methods.

Thirty-one Acinetobacter baumannii strains, comprising 14 strains from 14 outbreaks in different northwestern European cities and 17 sporadic strains, were compared by investigating various properties of the strains including biotype, antibiogram, cell envelope protein electrophoretic profile, ribotype pattern, and the band pattern generated by a novel genomic fingerprinting method, named AFLP, which is based on the selective amplification of restriction fragments. Results showed that 12 strains from unrelated outbreaks were linked together in two clusters according to their similarities by these typing methods, whereas sporadic strains were more heterogeneous. Outbreak strains appeared to be markedly more resistant to antibiotics than nonoutbreak strains. The uniformity of typing characters in two sets of outbreak strains suggests that strains in each cluster have a common clonal origin.

Acinetobacter↗
PubMed1995

[Risk factors of listeriosis in Denmark 1989-1990].

Risk factors for listeriosis are foods and underlying diseases. A case-control-study of listeriosis patients' dietary habits showed that unpasteurized milk was a risk food for sporadic listeriosis. A blue-mould cheese was significantly more often eaten by patients ill with an epidemic phage type. Leukaemia, AIDS and renal transplantation were found to be associated with a more than 1000 times higher risk of acquiring listeriosis, compared with healthy persons. As a part of prophylaxis, persons with a high risk of listeriosis should be informed individually about food hygiene and risk foods.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Diversity of nosocomial Xanthomonas maltophilia (Stenotrophomonas maltophilia) as determined by ribotyping.

Seventy-seven clinical isolates of Xanthomonas maltophilia (Stenotrophomonas maltophilia) were consecutively collected from the Rigshospitalet, Copenhagen, ribotyped and compared with the ribotypes of 25 other clinical and reference strains of Xanthomonas maltophilia. Using restriction enzyme EcoRI, 20 different ribotypes were observed, with 78 isolates displaying the five most common types. Using another enzyme, BamHI, these 78 isolates were further subdivided into 16 different ribotypes. Three patients harboured two strains with different ribotypes. No type was found related to only one department and no single-strain outbreak was detected. The origin of this wealth of different strains in hospital patients needs to be established.

Bacterial Typing Techniques↗