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Fawzy, A., M. Zschöck, C. Ewers, and T. Eisenberg, "Development of a hierarchical typing approach for Mycobacterium avium subsp. paratuberculosis (MAP) and characterization of MAP field cultures from Central Germany", Journal of applied microbiology, vol. 129, issue 5, pp. 1193 - 1206, 2020. Abstract

AIMSDevelopment of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany.

METHODS AND RESULTS

By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds.

CONCLUSIONS

The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time.

SIGNIFICANCE AND IMPACT OF THE STUDY

Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.

Fawzy, A., A. Fayed, H. Youssef, A. El-Sayed, and M. Zschöck, "First report of MIRU-VNTR genotyping of Mycobacterium avium subsp. paratuberculosis isolates from Egypt.", Iranian journal of veterinary research, vol. 17, issue 2, pp. 130-133, 2016 Spring. Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, an economically important disease in ruminants worldwide. It was first isolated in Egypt in 2005. Since then, the pathogen has been detected in different Egyptian provinces. In order to trace the source of infection, genotyping using simple methods of high discriminatory power such as mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR) were carried out in different countries. Until now there is no published information about MIRU-VNTR genotyping of MAP isolates in Egypt. To address that point, 100 faecal samples were collected and cultivated from 3 different suspected dairy farms. Fourteen isolates belonging to one farm were identified as MAP and subjected to genotyping using 8 different MIRU-VNTR loci PCRs. Two different genotypes were recognized based on size polymorphism observed in one locus (VNTR-7) that was confirmed by sequencing. Our work provides a preliminary basis of constructing a MIRU-VNTR genotyping database of MAP in Egypt.

Fawzy, A., M. Zschoeck, C. Ewers, and T. Eisneberg, "Genotyping methods and molecular epidemiology of Mycobacterium avium subsp. paratuberculosis (MAP)", Int J Vet Sci Med, vol. 6, issue 2, pp. 258-264, 2018. map_genotyping.pdf
Fawzy, A., A. - S. Giel, L. Fenske, A. Bach, C. Herden, K. Engel, E. Heuser, M. Boelhauve, R. G. Ulrich, K. Vogel, et al., "Development and validation of a triplex real-time qPCR for sensitive detection and quantification of major rat bite fever pathogen Streptobacillus moniliformis.", Journal of microbiological methods, vol. 199, pp. 106525, 2022. Abstract

Streptobacillus (S.) moniliformis is the most important pathogen causing rat bite fever (RBF) worldwide. This zoonotic pathogen is understudied mainly due to difficulties in culturing S. moniliformis as a fastidious microorganism. Therefore, advances in molecular detection techniques are highly needed, especially with regard to the widespread availability of real-time quantitative (q) PCR in laboratories. In this study, we aimed to develop a qPCR for the identification of Streptobacillus species and quantification of S. moniliformis in clinical samples, especially those derived from tissue samples of animal origin. We optimized a previously described PCR protocol in order to develop a qPCR, which can detect different Streptobacillus species with high specificity and is simultaneously able to quantitate S. moniliformis in different clinical matrices. The qPCR exhibited a limit of detection (LOD) of 21 copies/reaction representing ~4-5 streptobacilli, while the limit of quantification (LOQ) was 2.1 × 10 copies/reaction. It was also more sensitive than conventional PCR by two orders of magnitude and proved to have a substantial agreement (Kappa 0.74) compared to it with a superior detection rate in 374 samples from wild rats, laboratory rats and animals from holdings of wild-trapped rats. To conclude, the qPCR described in this study is an important molecular tool that is able to quantify S. moniliformis in tissue samples of animal origin. It represents a suitable tool for future establishment and evaluation of other molecular assays that are highly needed for a better understanding of epidemiology and pathophysiology of RBF. In experimental studies, it will also be useful for titration purposes since the quantification of the organism using classical plate counting technique is problematic and inaccurate.

Fawzy, A., J. Rau, K. Riße, N. Schauerte, C. Geiger, J. Blom, C. Imirzalioglu, J. Falgenhauer, A. Bach, C. Herden, et al., "Streptobacillus felis, a member of the oropharynx microbiota of the Felidae, isolated from a tropical rusty-spotted cat", Antonie van Leeuwenhoek, vol. 113, issue 10, pp. 1455 - 1465, 2020. Abstract

Streptobacillus felis is a fastidious microorganism and a novel member of the potentially zoonotic bacteria causing rat bite fever. Since its description, this is the second isolation of S. felis in a diseased member of the Felidae. Interestingly, the strain from this study was isolated from a zoo held, rusty-spotted cat (Prionailurus rubiginosus), with pneumonia, thereby indicating a possible broader host range in feline species. A recent preliminary sampling of domestic cats (Felis silvestris forma catus) revealed that this microorganism is common in the oropharynx, suggesting that S. felis is a member of their normal microbiota. Due to unawareness, fastidiousness, antibiotic sensitivity and lack of diagnostics the role of S. felis as a cat and human pathogen might be under-reported as with other Streptobacillus infections. More studies are necessary to elucidate the role of S. felis in domestic cats and other Felidae in order to better estimate its zoonotic potential.

Fawzy, A., M. Zschöck, C. Ewers, and T. Eisenberg, "New polymorphisms within the variable number tandem repeat (VNTR) 7 locus of Mycobacterium avium subsp. paratuberculosis.", Molecular and cellular probes, vol. 30, issue 3, pp. 132-7, 2016 Jun. Abstract

Variable number tandem repeat (VNTR) is a frequently employed typing method of Mycobacterium avium paratuberculosis (MAP) isolates. Based on whole genome sequencing in a previous study, allelic diversity at some VNTR loci seems to over- or under-estimate the actual phylogenetic variance among isolates. Interestingly, two closely related isolates on one farm showed polymorphism at the VNTR 7 locus, raising concerns about the misleading role that it might play in genotyping. We aimed to investigate the underlying basis of VNTR 7-polymorphism by analyzing sequence data for published genomes and field isolates of MAP and other M. avium complex (MAC) members. In contrast to MAP strains from cattle, strains from sheep displayed an "imperfect" repeat within VNTR 7, which was identical to respective allele types in other MAC genomes. Subspecies- and strain-specific single nucleotide polymorphisms (SNPs) and two novel (16 and 56 bp) repeats were detected. Given the combination of the three existing repeats, there are at least five different patterns for VNTR 7. The present findings highlight a higher polymorphism and probable instability of VNTR 7 locus that needs to be considered and challenged in future studies. Until then, sequencing of this locus in future studies is important to correctly assign the underlying allele types.(1).

Fenske, L., I. Noll, J. Blom, C. Ewers, T. Semmler, A. Fawzy, and T. Eisenberg, "A dominant clonal lineage of Streptococcus uberis in cattle in Germany.", Antonie van Leeuwenhoek, vol. 115, issue 7, pp. 857-870, 2022. Abstract

Bovine mastitis causes enormous economic losses in the dairy industry with Streptococcus uberis as one of the most common bacterial pathogens causing clinical and subclinical variations. In most cases mastitis can be cured by intramammary administration of antimicrobial agents. However, the severity of the clinical manifestations can vary greatly from mild to severe symtoms. In this study, a comparative genomic analysis of 24 S. uberis isolates from three dairy farms in Germany, affected by different courses of infection was conducted. While there were sporadic mild infections in farm A and B, a large number of infections were observed within a very short period of time in farm C. The comparison of virulence genes, antimicrobial resistance genes and prophage regions revealed no features that might be responsible for this severe course. However, almost all isolates from farm C showed the same, novel MLST profile (ST1373), thus a clonal outbreak cannot be excluded, whereby the actual reason for the particular virulence remains unknown. This study demonstrates the importance of extensive metagenomic studies, including the host genomes and the environment, to gain further evidence on the pathogenicity of S. uberis.