Publication Database

In silico genome analysis and molecular typing of Clostridium perfringens (2019)

Art

Hochschulschrift

Autor

Abdel-Glil, Mostafa Y. (WE 7)

Quelle

Berlin: Mensch & Buch Verlag Berlin, 2019 — V, 163 Seiten

ISBN: 978-3-96729-030-1

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/26585

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51843 / 66949
mikrobiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Main objectives of the thesis: 1. In silico investigation of the genomic variability, phylogenetic relatedness and virulence assessment of C. perfringens by means of comparative genome analysis employing publically available genomic data. 2. Isolation, characterization and genotyping of C. perfringens strains from healthy and diseased poultry collected from different farms and slaughterhouses in Egypt. 3. Development and application of a core genome-based multilocus sequence typing system for C. perfringens. 1. In silico analysis of the genomic variability, phylogenetic relatedness and virulence genes of C. perfringens The Gram-positive anaerobic spore forming bacterium C. perfringens is able to produce a large number of toxins by which it can cause various defined diseases in different hosts. With the aim to investigate its genomic diversity the publically available genome sequence data of 76 C. perfringens strains from diverse ecological, geographical and temporal niches were analyzed. Data analysis included 30 complete genomes which were composed of a circular chromosome (2.9 to 3.5Mbp) and up to six extrachromosomal elements. A substantial degree of genomic variability was detected in respect to episome content, chromosome size and mobile elements. Insertion sequences were identified and revealed abundance of their occurrence in certain genomes. Comparative alignment of complete genomes displayed a considerable degree of conservation in the order of genes in each chromosome except for three (out of the 30) genomes. The analyzed 76 C. perfringens strains were divided into three different phylogroups (I - III). Phylogroup I consisted of human food poisoning strains with chromosomal cpe as well as a Darmbrand (enteritis necroticans) strain. This phylogroup is characterized by a significant enrichment in mobile elements, relative small genome size and marked loss of chromosomal genes. Phylogroup I strains lack also two putative iron uptake systems as well as the pfoA gene. The genomic features of this phylogroup I (abundance of IS elements and genome reduction) provide indications that these strains adapt to a certain habitat causing human foodborne illnesses. Also, the absence of certain virulence genes (iron uptake systems and PFO) indicates the strains’ adaptation to less competitive environment (food) for replication. The loss of chromosomal genes in phylogroup I was in contrast to phylogroup II, in which the genome size indicates the addition of new genetic material. Phylogroup II strains carry also an additional putative iron uptake operon and an additional copy of the pfoA gene. Strains of this phylogroup were frequently reported in different animal hosts (equine and canine) in which they can cause enteric lesions. In sum, this study provides new insights into genomic variability and phylogenetic structure of C. perfringens. Phylogroup I (chromosomal cpe and Darmbrand strains) appears to be exposed to certain evolutionary mechanisms and displays characteristics that indicate speciation of these strains 2. Characterization and typing of C. perfringens isolates from healthy and diseased poultry in Egypt To investigate the diversity of poultry strains of C. perfringens isolated from Egypt, 54 birds from 27 farms suspected for necrotic enteritis (NE) as well as 50 healthy birds (10 ducks, 40 chickens) from eight slaughterhouses were sampled. C. perfringens was isolated from birds suspected of NE in 14 different farms (n = 51 isolates) as well as from apparently healthy poultry at slaughterhouses (n = 83 isolates). The C. perfringens isolates from suspected NE cases in Egypt were of toxin type A and netB negative, despite fact that NetB toxin was reported to play an important role in NE pathogenesis. The beta2 toxin gene was detected in both diseased and non-diseased birds. Ten isolates from five healthy ducks that did not produce the typical dual hemolysis on blood agar were identified. In addition, seven isolates from two cases suspected for NE were detected with an insertion of 834bp DNA segment within the amplicon of alpha toxin gene. The inserted DNA segment was identical to group II intron. C. perfringens strains that carry the group II intron were reported previously in Japan, Italy and Denmark. Interestingly, the insertion was detected so far within chicken isolates only. Additionally, a pilot investigation based on classical MLST was performed to determine the genetic relatedness of C. perfringens isolated in individual diseased birds. The investigated isolates of two birds (bird no. 1 and no. 2) belong to a single MLST sequence type each (ST45 and ST46) in concordance with various reports that described limited diversity of strains in NE affected birds. However, the isolates from bird no. 3 belonged to three different STs. Comparing MLST data for the 12 strains of this study and MLST data described in prior investigations revealed that the 12 strains were assigned to new STs and do not belong to the previously described “NE-associated” genotypes (ST31, CC-4). 3. Development and application of a core genome-based multilocus sequence typing system for C. perfringens Whole genome sequencing can provide a complete overview on organism genetic information but also represents a powerful molecular epidemiological tool for pathogen subtyping and outbreak investigations. In this study, a cgMLST scheme of 1,450 genes was developed for C. perfringens typing. The developed scheme was applied on a set of 160 genomes. An average of 99.5% of the cgMLST targets was found typeable per each genome. This scheme has a greater discriminatory power than the classical MLSTs methods. In addition, a whole genome based SNP typing was performed. The discriminatory power between the cgMLST and the whole genome SNP typing was comparable. The developed cgMLST scheme was applied using a cluster type (CT) threshold of 60 allelic differences to analyze 87 genomes of poultry strains of C. perfringens. Based on cgMLST results, most CTs comprised isolates derived from a single country only. However, few CTs harbor strains which were isolated in different countries. This could be due to the poultry commercial system which is likely maintained by few companies i.e. few sources worldwide. Compared to isolates from healthy birds and meat samples, a limited diversity was found in the suspected necrotic enteritis (NE) isolates from Egypt, supporting the hypothesis that distinct isolates cause NE. Isolates from diseased birds were found to group with isolates from healthy birds or meat samples highlighting the wide distribution of potentially virulent strains and the multifactorial character of the disease. Additionally, the 160 genomes investigated in this study were divided into four main phylogroups by hierBAPS. Whole genome SNP typing showed a superior applicability to delineate these phylogroups. A minimum SNP difference of ~ 40,000 SNPs was observed between the phylogroups. In sum, a useful cgMLST scheme for C. perfringens was developed that is applicable for broad and standardized epidemiological investigations. On the other hand, whole genome SNP typing can map the affiliation of individual isolates to the main phylogroups of C. perfringens in more detail.