Research

2022 – 2023: Development of a LAMP method for the detection of the novel species Trueperella pectoris

Trueperella pecoris is a new pathogen first described in 2021. The species was isolated in connection with mastitis in cattle, bronchopneumonia in pigs and reproductive tract diseases in various species. It is particularly problematic that the pathogenic significance of new pathogens can only be inadequately assessed, since the recognition of epidemiological correlations requires reliable diagnostics. However, detection methods for previously unknown species are usually not available. The development of molecular diagnostic techniques, such as a specific loop-mediated isothermal amplification (LAMP) assay, can provide a solution. This method, which is based on the amplification of nucleic acids, can be established in a short time and enables the rapid, sensitive and specific detection of bacterial pathogens. A particular advantage is that the reaction can be carried out inexpensively with a minimum of equipment, requires little time and works even with complex matrices without time-consuming DNA extraction. As part of a research project to develop a detection method for Trueperella pecoris, the pathogen could be reliably detected in lung tissue using the newly established LAMP assay.

Literature

Kreitlow, A., Ningrum, S. G., Lämmler, C., Erhard, M., Hoffmann, C., Plötz, M., Abdulmawjood, A. (2023). Identification of the novel potential pathogen Trueperella pecoris with interspecies significance by LAMP diagnostics. Scientific Reports, 27;13(1):14005.DOI: https://www.doi.org/10.1038/s41598-023-40787-1

2019 – 2022: Development of loop-mediated isothermal amplification (LAMP) assays for rapid detection of pathogens involved in foodborne infections and intoxications (2019 – 2022)

This research project is focused on developing and validating LAMP assays for the detection of Salmonella spp., Listeria monocytogenes, Bacillus cereus, Campylobacter jejuni, Campylobacter coli and Staphylococcus aureus. All species are food-associated zoonotic pathogens that can cause acute, self-limiting gastroenteritis, but sometimes also lead to severe intestinal and extraintestinal sequelae.

The LAMP method is used to identify the pathogens by amplification of specific DNA sequences. Six primers bind to a selected region within the pathogen genome and initiate sequence amplification under isothermal conditions. The use of a dye that intercalates with the LAMP products produces a fluorescent signal that can be measured using optical instruments (e.g., Genie® II real-time fluorometer). The performance of the assays is first tested using pure DNA and thereafter evaluated in complex matrices from various foods containing defined amounts of the pathogens. Finally, the assays are validated by testing naturally contaminated food samples from retail outlets and will then be available for analysis in the laboratory and further investigations in future research.

Literature

Kreitlow, A., Becker, A., Schotte, U., Malorny, B., Plötz, M., & Abdulmawjood, A. (2021). Establishment and validation of a loop-mediated isothermal amplification (LAMP) assay targeting the ttrRSBCA locus for rapid detection of Salmonella spp. in food. Food Control, 126, 107973.DOI: https://doi.org/10.1016/j.foodcont.2021.107973

Kreitlow, A., Becker, A., Schotte, U., Malorny, B., Plötz, M., & Abdulmawjood, A. (2021). Evaluation of different target genes for the detection of Salmonella sp. by loop‐mediated isothermal amplification. Letters in Applied Microbiology, 72(4), 420-426.https://doi.org/10.1111/lam.13409

Kreitlow, A., Becker, A., Ahmed, M. F., Kittler, S., Schotte, U., Plötz, M., & Abdulmawjood, A. (2021). Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products. Frontiers in microbiology, 12.DOI: https://doi.org/10.3389/fmicb.2021.668824

Busch, A., Becker, A., Schotte, U., Plötz, M., & Abdulmawjood, A. (2022). Mpl-Gene-Based Loop-Mediated Isothermal Amplification Assay for Specific and Rapid Detection of Listeria monocytogenes in Various Food Samples. Foodborne Pathogens and Disease.DOI: https://doi.org/10.1089/fpd.2021.0080

Busch, A., Schotte, U., Jeßberger, N., Frentzel, H., Plötz, M., Abdulmawjood, A. (2022). Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin Production. Frontiers in Microbiology. 9;13:930648.DOI: https://www.doi.org/10.3389/fmicb.2022.930648

2022 – 2024: Development of quantitative PCR assays for the detection of spoilage indicator organisms

Several real-time PCR assays are being developed as part of the project „Possibilities and limits of reducing salt and nitrite in raw sausages“ of the Research Association of the German Food Industry (FEI) and in cooperation with the German Institute of Food Technologies (DIL). These enable the rapid, specific and quantitative detection of certain bacteria involved in the spoilage of meat products. During challenge tests with bacterially inoculated products, the newly established qPCR assays can determine the growth behaviour of the pathogens throughout the storage period. This provides important information on the microbial stability and shelf life of the products. As a conventional real-time PCR assay cannot distinguish between living and dead cells, a propidium monoazide treatment is also to be established. This dye binds irreversibly to the DNA of cells with damaged membranes, enabling the differentiation between live and dead cells.

2022 – 2024: Development of a quantitative PCR method as a monitoring tool for hepatitis E virus contamination in the process chain

The molecular diagnostics of hepatitis E virus (HEV) in foodstuffs presents a number of challenges. The detection procedures must be capable of identifying the slightest traces of the virus, given that HEV does not replicate in foodstuffs. A significant challenge for molecular diagnostics is the highly heterogeneous nature of the virus genome. This presents a significant challenge for the development of specific primers, which are the foundation for a reliable detection of various HEV genotypes. The development and optimisation of a quantitative PCR assay, which will be evaluated in light of the aforementioned issues, is being conducted as part of a project funded by the Fritz-Ahrberg-Stiftung. Furthermore, the procedure will facilitate the first qPCR-based quantification of viral material in foodstuffs. In a second phase of the project, the method will be employed to monitor the contamination levels of foodstuffs along the production chain and to investigate potential reduction strategies for HEV contamination during production.

Literature

Hinrichs, J. B., Kreitlow, A., Plötz, M., Schotte, U., Becher, P., Gremmel, N., Stephan, R., Kemper, N., & Abdulmawjood, A. (2024). Development of a Sensitive and Specific Quantitative RT-qPCR Method for the Detection of Hepatitis E Virus Genotype 3 in Porcine Liver and Foodstuff. Foods (Basel, Switzerland), 13(3), 467. https://doi.org/10.3390/foods13030467

Hinrichs, J. B., Kreitlow, A., Siekmann, L., Plötz, M., Kemper, N., & Abdulmawjood, A. (2024). Changes in Hepatitis E Virus Contamination during the Production of Liver Sausage from Naturally Contaminated Pig Liver and the Potential of Individual Production Parameters to Reduce Hepatitis E Virus Contamination in the Processing Chain. Pathogens (Basel, Switzerland), 13(4), 274. https://doi.org/10.3390/pathogens13040274

There is an immense variety of different bacterial microorganisms in the world. In the Foodborne Zoonoses department, we regularly analyse isolates of previously unknown species. The characterisation of novel, potential pathogens includes a comprehensive analysis of biochemical and genotypic characteristics and thus contributes to the understanding of bacterial diversity, their habitats and potential hosts. In co-operation with other research institutions, we also publish genome data of unknown isolates and can define novel bacterial species on this basis.

Our most recently discovered species is called Arcanobacterium buesumense.

In addition, we are investigating in which animal hosts or other matrices recently discovered species occur in order to gain possible indications of their pathogenic potential and possible pathogen-host interactions. The most recently investigated pathogen candidates are

• Arcanobacterium canis
• Actinomyces weissii
• Arcanobacterium pinnipediorum
• Arcanobacterium phocisimile
• Arcanobacterium bovis
• Arcanobacterium wilhelmae

Literature

(2024). Complete genome sequence of Arcanobacterium wilhelmae strain DSM 102162 isolated from the genital tract of a Rhinoceros unicornis. Microbiology resource announcements, 13(8), e0020424. https://doi.org/10.1128/mra.00204-24

Borowiak, M., Kreitlow, A., Malorny, B., Lämmler, C., Prenger-Berninghoff, E., Plötz, M., & Abdulmawjood, A. (2024). Arcanobacterium canis strain DSM 25104 isolated from an English bulldog suffering from otitis externa: complete genome sequence. Microbiology resource announcements, 13(1), e0062423. https://doi.org/10.1128/mra.00624-23

Borowiak, M., Kreitlow, A., Malorny, B., Alssahen, M., Lämmler, C., Prenger-Berninghoff, E., Ewers, C., Siebert, U., Plötz, M., & Abdulmawjood, A. (2023). Arcanobacterium pinnipediorum Strain DSM 28752 Isolated from a Harbour Seal: Complete Genome Sequence. Microbiology resource announcements, 12(1), e0118022. https://doi.org/10.1128/mra.01180-22

Alssahen, M., Kreitlow, A., Sammra, O., Lämmler, C., Borowiak, M., Malorny, B., Siebert, U., Wohlsein, P., Prenger-Berninghoff, E., Plötz, M., & Abdulmawjood, A. (2022). Arcanobacterium buesumense sp. nov., isolated from an anal swab of a male harbour seal (Phoca vitulina). International journal of systematic and evolutionary microbiology, 72(10), 10.1099/ijsem.0.005573. https://doi.org/10.1099/ijsem.0.005573

Hijazin, M., Sammra, O., Ülbegi-Mohyla, H., Nagib, S., Alber, J., Lämmler, C., Kämpfer, P., Glaeser, S. P., Busse, H. J., Kassmannhuber, J., Prenger-Berninghoff, E., Weiss, R., Siebert, U., Hassan, A. A., Abdulmawjood, A., & Zschöck, M. (2013). Arcanobacterium phocisimile sp. nov., isolated from harbour seals. International journal of systematic and evolutionary microbiology, 63(Pt 6), 2019–2024. https://doi.org/10.1099/ijs.0.045591-0