Modules for Escherichia species#

--preset escherichia

These modules will be deployed if the enterobacterales__species module confirms the input assembly as a member of the Escherichia genus.

MLST#

-m escherichia__mlst_achtman, escherichia__mlst_pasteur

The Achtman scheme is hosted on EnteroBase and described in this paper.

We also provide an option for users to run MLST using the Pasteur scheme by running: -m escherichia__mlst_pasteur

The Pasteur scheme is hosted in the BIGSdb-Pasteur Escherichia coli database and described in this paper.

The genes included in each scheme are noted in the Outputs table below.

A copy of the MLST alleles and ST definitions used in each module is stored in the data/ directory of the module.

Parameters#

--escherichia_mlst_achtman_min_identity

Minimum alignment percent identity for Escherichia-Achtman MLST (default: 90.0)

--escherichia_mlst_achtman_min_coverage

Minimum alignment percent coverage for Escherichia-Achtman MLST (default: 80.0)

--escherichia_mlst_achtman_required_exact_matches

Minimum number of exact allele matches required to call an ST (default: 3).

--escherichia_mlst_pasteur_min_identity

Minimum alignment percent identity for Escherichia-Pasteur MLST (default: 90.0)

--escherichia_mlst_pasteur_min_coverage

Minimum alignment percent coverage for Escherichia-Pasteur MLST (default: 80.0)

--escherichia_mlst_pasteur_required_exact_matches

Minimum number of exact allele matches required to call an ST (default: 4).

Outputs#

Output of the Achtman E. coli MLST module include the following columns:

Column name

Content

ST_Achtman

Sequence type

adk, fumC, gyrB, icd, mdh, purA, recA

Allele numbers for the Achtman scheme loci

Output of the Pasteur E. coli MLST module includes the following columns:

Column name

Content

ST_Pasteur

Sequence type.

dinB, icdA, pabB, polB, putP, trpA, trpB, uidA

Allele numbers for the Pasteur scheme loci.

Notes#

  • Kleborate attempts to report the closest matching ST if a precise match is not found.

  • Imprecise allele matches are indicated with a *.

  • Imprecise ST calls are indicated with -nLV, where n indicates the number of loci that differ from the ST reported. For example, 131-1LV indicates a single-locus variant (SLV) of ST131, i.e. 6/7 loci match ST131.

cgMLST and LIN codes#

-m escherichia__cgMLST

This module performs cgMLST and LIN code typing using MiST tool.

Outputs#

cgMLST results are output in the following columns:

Column name

Content

cgST

Best matching scgST

LIN code

LIN code / Partial LINcode for input strain

Phylogroups#

-m escherichia__ezclermont

The Escherichia genus comprises several clades, including Escherichia albertii, E. fergusonii, five cryptic Escherichia clades (I–V) and E. coli sensu stricto. Within E. coli, strains can be further divided into seven main phylogroups: A, B1, B2, C, D, E and F.

Kleborate assigns Escherichia genomes to these clades and phylogroups using EzClermont tool, which is based on in vitro PCR assay logic.

Parameters#

--escherichia__ezclermont_min_length

Minimum contig length to consider. Default: 500

Outputs#

Column name

Content

Clermont_type

Assigned phylogroup or clade.

Clermont_profile

Presence or absence pattern of PCR products.

Pathotyping#

-m escherichia__pathovar

Escherichia coli is broadly divided into 2 groups: intestinal diarrheagenic E. coli (DEC), and extra-intestinal E. coli (ExPEC). DEC encompasses several clinically relevant pathotypes: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) see paper. Additionally, Shigella is considered a DEC pathotype due to its genetic and pathogenetic similarity to EIEC, see paper.

The majority of DEC pathotypes are defined by specific virulence markers. However, for EAEC, DAEC and AIEC, the pathogenic role of proposed markers is not well established.

This module classifies E. coli genomes into DEC pathotypes based on the presence or absence of key virulence marker genes, determined by alignment with Minimap2 to reference sequences for key marker genes, extracted from the VirulenceFinder database.

Marker genes considered are:

Gene

Marker of

ipaH

pINV plasmid shared by Shigella and EIEC

sta1 and ltcA

ST, heat-stable enterotoxin and LT, heat-labile enterotoxin, characteristic of ETEC

stx1A, stx1B, stx2A, stx2B

shiga-toxins Stx1 and Stx2

bfpA

bfp adhesin characteristic of EPEC

eae

intimin, characteristic of EPEC and EHEC

The module also calls ShigaPass to predict Shigella serotypes, and to differentiate Shigella from enteroinvasive Escherichia coli (EIEC).

The combination of marker genes detected are then used to classify genomes into pathotypes, using logic adapted from EnteroBase as follows:

Pathotype

Marker/s Present

Marker/s Absent

EIEC

pINV

ST and LT, Shigella serotypes

ETEC

ST or LT

pINV

STEC

Stx1 or Stx2

intimin

EHEC

Stx1 or Stx2, and intimin

EPEC

intimin and bfp

Stx1 and Stx2

aEPEC

intimin

Stx1 and Stx2 and bfp

Shigella

Shigella serotypes

Parameters#

--escherichia__pathovar_min_identity

Minimum alignment percent identity for pathotype (default: 90.0).

--escherichia__pathovar_min_coverage

Minimum alignment percent coverage for pathotype (default: 80.0).

Outputs#

Column name

Content

Pathotype

Predicted pathotype

Stx1, Stx2, ST, LT, eae, ipaH

Virulence markers

Additionally, Kleborate includes a separate escherichia__vfdb module that screens E. coli genome assemblies against all markers in the VirulenceFinder database (VFDB).

LEE pathogenicity island typing#

-m escherichia__mlst_lee

Locus of enterocyte effacement (LEE) is a ~40 kb chromosomal pathogenicity island composed of 41 core genes organized into five operons Elliot et al., 1998. It encodes an (i) outer membrane adhesive protein, known as intimin protein and encoded by the _eae_ gene; (ii) type III secretion system (T3SS), and (iii) translocated receptor (Tir) as well as translocons, chaperones, regulators and secreted effector proteins that are linked to virulence.

Kleborate includes a module for subtyping of the LEE pathogenicity island. Details of the the scheme, and the LEE subtypes and lineages it identifies, can be found in this paper.

The LEE typing database is based on analysis of >250 LEE-containing E. coli genomes and includes 7 loci (eae (intimin), tir, espA, espB, espD, espH, espZ). The data is provided as a MLST-style database, in which combinations of alleles are assigned to a LEE subtype, to facilitate a common nomenclature for LEE subtypes. Each sequence in the database represents a cluster of closely related alleles that have been assigned to the same locus type. The LEE scheme includes three distinct lineages: Lineage 1 consists of LEE subtypes 1-2; Lineage 2 consists of LEE subtypes 3-8; Lineage 3 consists of LEE subtypes 9-30.

The reference sequences and MLST-style profile definitions are included in the data/ directory of this module.

Parameters#

--escherichia__mlst_LEE_min_identity

Minimum alignment percent identity for escherichia__mlst_LEE. Default: 90.0

--escherichia__mlst_LEE_min_coverage

Minimum alignment percent coverage for escherichia_mlst_LEE. Default: 80.0

escherichia__mlst_LEE_mlst_required_exact_matches

Minimum number of exact allele matches required to assign an ST. Default: 3

Outputs#

The output of the E. coli LEE MLST module includes the following columns:

Column name

Content

LEE_ST

Assigned LEE sequence type.

LEE_lineage

Lineage associated with the LEE ST.

LEE_eae, LEE_tir, LEE_espA, LEE_espB, LEE_espD, LEE_espH, LEE_espZ

Allele numbers for each LEE locus.

Additional Notes#

  • Kleborate attempts to report the closest matching ST if an exact match is not found.

  • Imprecise allele matches are indicated with a *.

  • Imprecise ST calls are indicated with -nLV, where n indicates the number of loci that disagree with the ST reported. For example, ST10-3LV indicates a three-locus variant (SLV) of ST10 (i.e. 4/7 loci match ST10).

Stx typing#

-m escherichia__stxtyper

Shiga toxins (Stxs) are key virulence factors of Stx-producing Escherichia coli (STEC). They are also found in Shigella dysenteriae 1. Stxs belong to the AB-type toxin family and are divided into two antigenically distinct groups: Stx1 and Stx2. Each group contains several variants/subtypes—six for Stx1 (a, b, c, d, e, f) and seven for Stx2 (a, b, c, d, e, f, and g) (Yano et al., 2023, Melton-Celsa, 2014). These toxins are encoded by lysogenic bacteriophages (Stx phage) and STEC strains may produce either single Stx subtype or a combination of subtypes.

This module will run StxTyper <https://doi.org/10.3390/microorganisms14081607> to determine the _stx_ type. See the StxTyper documentation for more details of how it works.

Outputs#

StxTyper results are output in the following columns:

Column name

Content

Stx_type

The Shiga toxin type. If the operon is complete, the subtype will be reported (e.g., stx1a). If the operon is incomplete or ambiguous, a broader designation is used: stx1, stx2, or simply stx if the algorithm cannot resolve at further.

operon

Status the operon detected. Possible values: COMPLETE – Full operon found. PARTIAL – Operon incomplete. PARTIAL_CONTIG_END – Partial operon likely truncated at contig boundary. EXTENDED – Coding sequence extends beyond the reference stop codon for one or both subunits. INTERNAL_STOP – A subunit contains a nonsense mutation. FRAMESHIFT – Indel detected in coding sequence. AMBIGUOUS – Ambiguous base(s) found in the sequence. COMPLETE_NOVEL – Full-length operon that cannot be typed.

identity

Percent identity for both A and B subunits.

target_start

Start position of the alignment.

target_stop

End position of the alignment.

target_strand

Strand orientation of the target sequence.

A_reference

Closest reference protein for the A subunit.

A_identity

Percent identity to the reference for the A subunit.

A_reference_subtype

Subtype assigned to the reference sequence for the A subunit.

A_coverage

Percentage of the A subunit reference sequence covered by the alignment.

B_reference

Closest reference protein for the B subunit.

B_reference_subtype

Subtype assigned to the reference sequence for the B subunit.

B_identity

Percent identity to the reference for the B subunit.

B_coverage

Percentage of the B subunit reference sequence covered by the alignment.

Polyketide synthetase (colibactin) detection#

-m escherichia__pks

This module screens for the presence or absence of clbB, gene in the colibactin polyketide synthase (PKS) biosynthesis gene cluster, by aligning genome assemblies against the clbB reference sequence.

Parameters#

--escherichia__pks_min_identity

Minimum alignment percent identity for detecting clbB (default: 90.0)

--escherichia__pks_min_coverage

Minimum alignment percent coverage for detecting clbB (default: 80.0)

Outputs#

Column name

Content

clbB

presence or absence of the gene

O:H serotyping#

-m escherichia__ectyper

E. coli serotypes are defined by combinations of O (lipopolysaccharide) and H (flagellar) antigens. Currently there are ~183 O-groups and 53 H-types that have been defined serologically Ørskov and Ørskov 1984.

O-antigen

The O-antigen is an integral component of the Lipopolysaccharide (LPS) found in the outer membrane of the bacteria. LPS comprises three components: lipid A, a core oligosaccharide, and the O-specific polysaccharide chain (O antigen). The O-antigen domain exhibits significant variability consisting of 10 to 25 repeating oligosaccharide units, with each unit containing two to seven sugar residues Liu et al., 2020. The genes responsible for synthesis of O-antigens are usually present as a gene cluster and are located between the two chromosomal housekeeping genes galF and gnd/ugd Iguchi et al 2014. Major pathways involved in the assembly, synthesis and transport of O-antigen include, the Wzy pathway the Wzx/Wzy-dependent pathway, encoded by the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes, and the ABC transporter pathway, encoded by wzm and wzt. These genes are ideal biomarkers for predicting O antigen types.

H antigens

H antigens (flagellar) are surface proteins composed of repeated molecules of the protein flagellin, which facilitate bacterial motility. These antigens are numbered from H1 to H56 (H13, H22, and H50 are not used) and are distinct from the O and K antigens. Flagellin is encoded by the fliC gene on the chromosomal locus or its homologues (non-fliC flagellin-coding genes such as flkA, fllA, and flmA). Of the 53 well known H antigen types, 44 are conferred by expression of the fliC gene, the remaining 9 H types are encoded by non-fliC flagellin genes. Specifically H3, H35, H36, H47,and H53 are encoded by flkA, H44 and H55 by fllA, H54 by flmA, and H17 by flnA.

Kleborate uses ECTyper for in silico serotyping. See ECTyper paper for more details.

Outputs#

Outputs of the ECTyper module is the following columns:

Column name

Content

O-type

Predicted O antigen.

H-type

Predicted H antigen.

Serotype

Combined prediction of O and H antigens.

QC

Quality control values summarising the overall confidence of the serotype prediction.

Evidence

Total number of alleles used to call both O and H antigens.

GeneScores

ECTyper gene scores for O and H antigens, ranging from 0 to 1.

AllelesKeys

Best-matching allele keys from the ECTyper database used for serotype assignment.

GeneIdentities(%)

Percent identity values of the query alleles.

GeneCoverages(%)

Percent coverage values for the query alleles.

GeneLengths

Gene lengths ( in base pairs) of the query alleles.

Warnings

Additional messages related to QC status or other issues affecting serotype prediction.

Capsule (K) typing#

-m escherichia__kaptive

This module will run the Kaptive v3 tool to type E. coli group 2 and group 3 capsular (K) loci. See the capsular K-typing database for more details.

Parameters#

--ecoli_kps

Group 2 + 3 CPS database

Outputs#

Kaptive results are output in the following columns:

Column name

Content

K_locus

The locus type which most closely matches the assembly.

K_type

The predicted serotype/phenotype of the assembly.

K_locus_confidence

Typeable or Untypeable.

K_locus_problems

Characters indicating issues with the locus match (see problems).

K_locus_identity

Weighted percent identity of the best matching locus to the assembly.

K_Missing_expected_genes

Missing expected genes in the locus

Antimicrobial resistance#

-m escherichia__amr

This module screens input genomes for acquired antimicrobial resistance genes and known resistance-associated point mutations using the AMRFinderPlus tool. Identified determinants are grouped by drug class.

Parameters#

--organism

Used to screen for point mutations in species-specific resistance markers (default Escherichia coli).

-t , --threads

Number of threads to use for alignment.

Outputs#

Results of the Escherichia AMR module are grouped by drug class:

Column name

Markers assigned as NCBI class/subclass

Penicillins

BETA-LACTAM, CEPHALOTHIN

Carbapenems

CARBAPENEM

ESBL

CEPHALOSPORIN (note these can be assumed to be 3rd generation cephalosporins)

Beta-lactamase inhibitor

TANIBORBACTAM, AMOXICILLIN-CLAVULANIC ACID, PIPERACILLIN-TAZOBACTAM, TICARCILLIN-CLAVULANIC ACID, SULBACTAM-DURLOBACTAM, CEFTAZIDIME-AVIBACTAM, TAZOBACTAM

Aminoglycosides

AMINOGLYCOSIDE

Quinolones

FLUOROQUINOLONE, QUINOLONE

Fosfomycin

FOSFOMYCIN

Colistin

COLISTIN

Phenicols

PHENICOL

Macrolides

MACROLIDE

Rifamycin

RIFAMYCIN

Sulfonamides

SULFONAMIDE

Tetracyclines

TETRACYCLINE

Tigecycline

TIGECYCLINE

Trimethoprim

TRIMETHOPRIM

Other Classes

other CLASS values