Escherichia coli diagnostics

Transcription

1 Escherichia coli diagnostics Workshop on Whole Genome Sequencing and Analysis, Mar. 2018

2 Learning objective: After this lecture and exercise, you should be able to describe how the CGE methods for identifying the serotype of E. coli, and virulence factors of E. coli, Enterococcus, Listeria, and S. aureus work use the above-mentioned methods as stand-alone services and interpret the results retrieve information about the genes in the CGE databases generate your own database in FASTA format and use it for searching for specific genes of interest

3 Escherichia coli pathogenicity E. coli is naturally found in the intestinal tract of humans and many other animals Most strains are harmless commensals Acquisition of combination of genetic mobile elements leads to pathogenic phenotype

4 Pathogenic E. coli are able to colonise many sites of the human body EHEC strains comprise a subgroup of Shiga-toxin (Stx)- producing E. coli and cause illness in humans

5 Simplified categorisation that we will use during exercise Non-pathogenic: Few or no obvious virulence factors. UPEC: Adhesion factors to avoid being flushed away from the urinary tract. Also siderophore proteins (e.g., encoded by iron). Presence of Pap (P) fimbriae (papg adhesion) can be a sign of increased virulence. EHEC (VTEC, STEC): The hallmark virulence factor is Shiga toxin (Stx). Stx consists of an A subunit (encoded by stxa) and B subunits (encoded by stxb). EAEC: Contains a 100-kb paa plasmid with many different virulence factors (especially aggregative adherence fimbriae (AFFs), mycolycins (e.g., encoded by pic), and toxins (e.g., encoded by pet and asta). A regulator encoded by the aggr gene, also located on the paa plasmid, is coordinating the virulence factors. ETEC: This pathotype is known for its production of Heat-Stabile Toxin (ST) or Heat-Labile Toxin (LT). The former can be encoded by the sta1 or stb genes and the latter by ltca.

6 Shiga toxin-producing E. coli (STEC) / Verocytotoxin-producing E. coli (VTEC) A gastrointestinal pathogen Produces verocytotoxins as well as other virulence factors May cause bloody diarrhoea and occasionally Haemolytic Uremic Syndrome (HUS) Primary sources are undercooked ground meet, raw milk, and faecal contamination of vegetables In EU, in 2013, there were 6,043 confirmed cases of VTEC and 13 deaths due to VTEC

7 Previous routine typing of VTEC by the Danish State Serum Institute Serotyping Examined for.... β-glucuronidase activity.. haemolysin production.. Verocytotoxin production.. Presence of specific virulence factors, most importantly, verotoxin 1 (vtx1), verotoxin 2 (vtx2) and intimin (eae), which were detected by DNA hybridization Further subtyping of the verocytotoxins was carried out by PCR Isolates with same serotype and toxin profile compared by PFGE

8 Serotyping In serotyping a group of closely related microorganisms are distinguished by a characteristic set of antigens by the use of antibodies E. coli SerotypeFinder O-type is based on the O-processing genes: wzx, wzy, wzm, and wzt, and wzx/wzy or wzm/wzt pairs H-type is based on the detection of flagellin genes: flic, flka, flla, flma, flna O:H serotyping of E. coli using WGS data Joensen KG. et al J.Clin.Microbiol. 53(8):

9 Evaluating the performance of SerotypeFinder Number of genomes for validation with detected genes with consistent WGS -and conventional results O-typing (~95%) 560 (~98%) H-typing (~100%) 503 (~99%) Phenotype vs. genotype - Rough, non-motile, non-typable gets a genotype, which is not the actual serotype Non-distinguishable: - O90/O127 - O107/O117 - O20/O137 - O13/O135/O129

10 Serotyping of Salmonella: SeqSero ( SalmonellaTypeFinder ( Serotyping of P. aeruginoa: PAst (

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12 VirulenceFinder Joensen et al J. Clin. Microbiol. 52(5):

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14 Gene content of the E. coli virulence database

15 Database content Static databases at are no longer maintained All databases are in an online repository called BitBucket BitBucket: Web-based hosting service aimed at software version control systems for teams Main advantages: It is easy to see which changes have been done since last update It is possible to roll back to previous versions

16 _db means it is a database repository Otherwise it is a software repository

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21 AU! Mouse arm SourceTree to the rescue!

22 Graphical User Interface client for Windows and Mac Used for connecting databases of interest stored on local computer with BitBucket repositories SourceTree makes it easy to see if changes have been made to BitBucket repository and to download (pull) those changes

23 BLAST-based CGE finder tools >Gene_1 GATAATGTAATAGAAATTGGATCAGGAAAAG GTCATTTTACCAAAGAACTTGTCAAAATGAG TCGGTGGGTGGATTCTATAGAAATTGATGAG GACTTGTGTCAAGTCACCCAAAAAGTCGTGA AGCCTTTTCAGAATATAAAAGTTATCCATAC GGATATTCTGAAATTTAACTTCCCCAAAAAC AAAGACTATAAAATATTTGGTAATATCCCCT TTAACATCAGTACTGATATTGTCAAAAAAAT TGCTTTTGAAAGCAATTCGAAATATAGTTA >Gene_2a ATGAACAAAAATATAAAATATTCTCAAAACT TTTTAACGAGTGAAAAAGTACTCAACCAAAT AATAAAACAATTGAATTTAAAAGAAACCGAT ACCGTTTACGAAATTGGAACAGGTAAAGGGC ATTTAACGACGAAACTGGCTAAAATAAGTAA ACAGGTAACGTCTATTGAATTAGACAGTCAT CTATTCAACTTATCGTCAGAAAAA Gene_2b ATGAAAAAAAATATAAAATATTCTCAAAACT TTTTAACGAATGAAAAGGTACTCAACCAAAT AATAAAACAATTGAATTTAAAAGAAACCGAT ACCGTTTACGAAATTGGAACAGGTAAAGGGC ATTTAACGACGAAACTGGCTAAAATAAGTAA ACAGGTAACGTCTATTGAATTAGACAGTCAT CTATTCAACTTATCTTCAGAAAAA Database >Contig_1 TGGATTCTATAGAAATTGATGAGGACTTGTG TCAAGTCACCCAAAAAGTCGTGAAGCCTTTT CAGAATATAAAAGTTATCCATACGGATATTC TGAAATTTAACTTCCCCAAAAACAAAGACTA TAAAATATTTGGTAATATCCCCTTTAACATC AGTACTGATATTGTCAAAAAAATTGCTTTTG AAAGCAATTCGAAATATAGTTAAATATAAAA TATTCTCAAAACTTTTTAACGAGTGAAAAAG TACTCAACCAAATAATAAAACAATTGAATTT AAAAGAAACCGATACCGTTTACGAAATTGGA ACAGGTAAAGGGCATTTAACGACGAAACTGG CTAAAATAAGTAAACAGGTAACGTCTATTGA ATTAGACAGTCATCTATTCAACTTATCGTCA GAAAAAATGAAAAAAAATATAAAATATTCTC AAAACTTTTTAACGAATGAAAAGGTACTCAA CCAAATAATAAAACAATTGAATTTAAAAGAA ACCGATACCGTTTACGAAATTGGAACAGGTA AAGGGCATTTAACGACGAAACTGGCTAAAAT AAGTAAACAGGTAACGTCTATTGAATTAGAC AGTCATCTATTCAACTTATCTTCAGAAAAA >Contig_12 AGTGTGTGCGAGAGAGCGAGCGTGCGTGCGA GAGAGTTTCGCGCGCGCTTTAGAGAGCGTGC GAGCGAGCGAGCGTGTTTGTGCCCCAGAGAA ATGGTAATATCCCCTTTAACATCAGTACTGA TATTGTCAAAAAAATTGCTTTTGAAAGCAAT TCGAAATATAGTTAAATATAAAATATTCTCA AAACTTTTTAACGAGTGAAAAAGTACTCAAC CAAATAATAAAACAATTGAATTTAAAAGAAA CCGATACCGTTTACGAAATTGGAACAGGTAA Draft assembly

24 BLAST-based CGE finder tools MLST ResFinder PlasmidFinder pmlst SerotypeFinder VirulenceFinder

25 Generating your own database Collect DNA sequences of the genes you wish to search for Save the DNA sequences in FASTA format Take care to provide each gene with a unique identifier (everything from the > to the first space) Save the FASTA file as pure text Congratulations - you have made your own database!

26 MyDBFinder Use your own database with any gene(s) of interest

27 Exercise 2 data

28 > Just one out of many papers about this outbreak

29 A single lot of fenugreek seeds, from an exporter in Egypt German farm French farm German outbreak Smaller outbreak in France: On June 24, 2011, France identified a cluster of E. coli O104:H4 infections among attendees of an event in Bordeaux, France

30 By July 2011 the outbreak was over: German outbreak: 4,000 cases of bloody diarrhoea 850 cases of HUS 50 deaths French outbreak: 15 cases of bloody diarrhoea 9 cases of HUS 0 deaths

31 Characterisation of the bacteria causing the outbreak Species: Escherichia coli Serotype: O104:H4 (historically, E. coli with this serotype is enteroaggregative, but not Shiga toxin-producing) MLST: ST-678 Plasmids: Name Size Note (kbp) pg-ea Cryptic plasmid, only encodes repa genes pesbml-ea11 90 IncI1 plasmid, carries CTX-M-15 beta-lactamase paa-ea11 75 a Tn5-insert derivative of adherence plasmid Ahmed S. et al 2012, PLOS One, 7 (11). e48228

32 Antibiotic resistance profile Resistance Ampicillin and 3rd gen. cephalosporins (ESBL production) Streptomycin Nalidixic acid Tetracyclin Cotrimoxazole Susceptible Carbapenems Ciprofloxacin Chloramphenicol Kanamycin Gentamicin It is not recommend that STEC infections are treated with antibiotics, so the fact that this strain is resistant towards certain types of antibiotics does not impact the care that infected patients receive