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Burkhard Malorny

Publications and source records attributed to Burkhard Malorny.

15 recordsLinked to original sources
PubMed2006

Molecular characterisation of Salmonella strains by an oligonucleotide multiprobe microarray.

A DNA microarray has been developed for the simultaneous characterisation and typing of Salmonella enterica subsp. enterica isolates. One-hundred and nine 35-40 mer oligonucleotides probes detect flagellar and somatic antigen encoding genes (serogroup or serotype specific), important virulence genes located within or outside the pathogenicity islands, phage-associated genes and antibiotic resistance determinants. The probes were printed on glass slides and whole genomic Cy5-labelled Salmonella DNA was hybridised to the substrate. A set of 19 different Salmonella strains and one Escherichia coli strain has been selected as positive and negative controls for each probe. The validity of the results is confirmed by gene-specific PCRs or phenotypic methods (serotyping, MIC determination for various antimicrobial agents). Of 2071 data points generated, an agreement of 97.4% has been obtained between microarray and PCR/phenotypic results. Twenty-six data points (1.3%) were classified as uncertain and, similarly, 1.3% showed a discordant result. The microarray described here is a new tool to study the epidemiology of Salmonella strains on the genotypic level and might become a powerful method in risk assessment studies.

Bacteriophages↗
PubMed2004

Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples.

In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancements in PCR technology, we have designed two specific PCR assays for detecting Salmonella spp. We have compared PCR-enzyme-linked immunosorbent assay (PCR-ELISA) and LightCycler real-time PCR assay (LC-PCR) with the standard ISO 6579 bacteriological reference method. The two PCR tests incorporated an internal amplification control (IAC) co-amplified with the invA gene of Salmonella to monitor potential PCR inhibitors and ensure successful amplification. The selectivity study involved 84 Salmonella and 44 non-Salmonella strains and the samples tested were represented by 60 artificially-contaminated samples of fish, minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3)CFU/ml, which corresponds respectively to 50 and 10 cells per PCR tube. Data on artificially contaminated samples indicated that both PCR methods were able to detect after enrichment less than five Salmonella cells in 25 g of food, giving 100% concordance with the ISO 6579 reference method. The results on naturally contaminated samples demonstrated that despite certain inhibition problems, LC-PCR and PCR-ELISA assays were highly specific and sensitive, and provide a powerful tool for detection of Salmonella in food samples.

Animals↗
PubMed2004

Diagnostic real-time PCR for detection of Salmonella in food.

A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

Animals↗
PubMed2003

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method.

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n = 285), whole broiler carcass-rinse (n = 25), various raw meat (n = 33), and environmental samples (n = 92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.

Animals↗
PubMed2003

Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry.

OBJECTIVE: Phenotypic and genotypic characterization of the antimicrobial resistance of German Escherichia coli strains isolated during 1999-2001 from cattle, swine and poultry. MATERIALS AND METHODS: Three hundred and seventeen isolates were tested for their resistance to 17 antimicrobial agents by broth microdilution. Resistant strains were screened by molecular methods for resistance genes, integrons and mutations in quinolone-resistance determining regions. RESULTS: Resistance was found in 40% and multiresistance in 32% of the strains. The resistance was significantly higher in isolates from poultry (61%) and swine (60%) than from cattle (25%) (P < 0.01). The most prevalent resistances were to sulfamethoxazole, tetracycline, streptomycin, ampicillin and spectinomycin (30-15%). For each antibiotic, the predominant resistance genes were: ampicillin, blaTEM1-like (92%); chloramphenicol, catA (68%) and cmlA1-like (36%); gentamicin, aac(3)-IV (60%); kanamycin, aphA1 (100%); streptomycin, aadA1-like (61%) and strA/B (59%); sulfamethoxazole, sul2 (66%), sul1 (42%) and sul3 (14%); tetracycline, tet(A) (66%) and tet(B) (42%); and trimethoprim, dfrA1-like (77%), dfrA17 (13%) and dfrA12 (7%). Class 1 integrons were found in 30% of the strains. They carried dfrA1-aadA1a (40%), aadA1a (29%), sat1-aadA1a (16%), dfrA17-aadA5 (11%), oxa1-aadA1a (5%) and dfrA12-aadA2 (3%). Eleven percent of the strains were resistant to nalidixic acid. Of these, 61% presented a reduced susceptibility to ciprofloxacin (MIC = 0.12-2 mg/L) and single mutations in gyrA or gyrA and parC genes, and 39%, full resistance to ciprofloxacin (MIC > or = 4 mg/L) and double and single mutations in gyrA and parC, respectively. CONCLUSION: The study gives baseline information on the magnitude of the resistance problem and its genetic background in contemporary German E. coli from food-producing animals.

Amino Acid Substitution↗
PubMed2003

Standardization of diagnostic PCR for the detection of foodborne pathogens.

In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection of pathogenic bacteria in foods. The present review focuses on the harmonization procedure and standardization criteria for detection of foodborne pathogens by PCR. The progress of standardization so far and future perspectives of diagnostic PCR are discussed.

Bacteria↗
PubMed2003

Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard.

As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific for Salmonella species were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43 Salmonella spp. and 47 non-Salmonella strains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets the invA gene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for the invA primer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with the invA target gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 10(4) CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded ("blind") DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific for Salmonella spp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.

Animals↗
PubMed2003

Discrimination of d-tartrate-fermenting and -nonfermenting Salmonella enterica subsp. enterica isolates by genotypic and phenotypic methods.

A multiplex PCR and an improved lead acetate test were developed to discriminate d-tartrate-fermenting and -nonfermenting Salmonella enterica subsp. enterica strains. Both methods showed an accuracy of 100% when 125 Salmonella strains belonging to 15 serovars were tested. Special emphasis was given to S. enterica subsp. enterica serovar Paratyphi B isolates because of the clinical importance of its d-tartrate-nonfermenting variant and the recently increasing numbers of cases of human outbreaks caused by its fermenting variant (formerly Salmonella serovar Java). The lead acetate test described previously (G. A. Alfredsson, R. M. Barker, D. C. Old, and J. P. Duguid, J. Hyg. 70:651-666, 1972) was modified in the inoculation and incubation procedure. The PCR assay was based on the genotypic difference of the presence (d-tartrate-fermenting strains) or absence (d-tartrate-nonfermenting strains) of the ATG start codon for the gene STM 3356, which encodes a putative cation transporter. Sequence data revealed a nucleotide exchange from G to A within the ATG start codon of gene STM 3356 in the d-tartrate-nonfermenting strains. In order to increase the reliability of the PCR assay, a positive control based on a Salmonella genus-specific primer set for the detection of Salmonella DNA was included. The PCR-based discrimination needs only several hours compared to 6 days needed by the improved lead acetate test to obtain reliable results. Consequently, the PCR d-tartrate assay should be the method of choice for the discrimination of d-tartrate-fermenting and -nonfermenting Salmonella strains in the future.

Base Sequence↗
PubMed2002

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction.

A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.

Animals↗
PubMed1998

Recombinational reassortment among opa genes from ET-37 complex Neisseria meningitidis isolates of diverse geographical origins.

Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. ET-37 complex meningococci, defined by multilocus enzyme electrophoresis, have been isolated on different continents. Twenty-six different Opa proteins have been observed within strains of the ET-37 complex isolated between the 1960s and the 1980s, although individual strains have only four opa genes per chromosome. In this work the opa genes of four closely related ET-37 complex N. meningitidis strains recently isolated from Mali, West Africa were characterized and compared with the opa genes of strain FAM18, an ET-37 complex isolate from the USA. DNA sequence analysis and Southern blot experiments indicated that recombinational reassortment, including gene duplication and import by horizontal genetic exchange, has occurred in the opa genes within the ET-37 complex, resulting in two partially different Opa repertoires being present in FAM18 and the Mali isolates. Using synthetic peptides derived from the hypervariable (HV) regions of opa genes, the epitopes for nine mAbs were mapped. These bacteria, isolated on different continents, contain both shared and unique opa HV regions encoding epitopes recognized by mAbs and show evidence of recombinational reassortment of the HV regions.

Amino Acid Sequence↗
PubMedDate not supplied

[Typing of Salmonella by DNA-microarrays].

Microarrays (DNA-Chips) are miniaturized carriers on which many nucleic acid molecule probes such as oligonucleotides or PCR products are immobilized in a high density, and compactness. Homologue DNA hybridises with the immobilized complementary nucleic acid probes. This study gives after a short general introduction in the principle of DNA-microarrays an overview about published data on the field of typing of Salmonella by microarrays. An onset of a DNA-microarray developed by the National Reference Laboratory for Salmonella (NRL-Salm) will be introduced. By this new technique, it is possible to answer epidemiological questions as well as to find genes involved in certain biochemical processes, such as pathogenicity or resistance of salmonellae.

Animals↗
PubMedDate not supplied

Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples.

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.

Animals↗