REFERENCE PROTOCOL

MLST Protocol for Klebsiella variicola

Primary Reference

Molecular epidemiology of Klebsiella variicola obtained from different sources

Scientific Reports 2019 DOI: 10.1038/s41598-019-46998-9

MLST Gene Targets

Gene	Function
leuS	Leucyl-tRNA synthetase
pgi	Phosphoglucose isomerase
pgk	Phosphoglycerate kinase
phoE	Phosphoporine E
pyrG	CTP synthase
rpoB	β-subunit of RNA polymerase B
fusA	Elongation factor G

PCR Amplification Protocol

Gene	Primer	Sequence (5'-3')	Temp (°C)	Size (bp)
leuS	F	GGCGTTGATATTGGCGATGG	60	933
leuS	R	GCCGCCGCCGCCGCCGCCAA	60	933
pgi	F	ATGGCTAAACTGGGTAAAGGTG	60	801
pgi	R	TTACGCCTGAACTGCTTGCC	60	801
pgk	F	ATGAAAAAATTATTGATTTTATTG	55	1242
pgk	R	TTATTTTTTCCGCCCTTCAC	55	1242
phoE	F	ATGAAAGTTTTAGTTATTATTG	55	906
phoE	R	TTATTTTTTAGCGCCGTTTC	55	906
pyrG	F	ATGGCGAAAGTTATTGTTG	55	1095
pyrG	R	TTATTTGCCGCCGCCGCCAA	55	1095
rpoB	F	ATGTCGCGTATTGAAGGTG	60	903
rpoB	R	TTACGCCTTGTTGCCGCCG	60	903
fusA	F	ATGGCGAAAGTTATTGTTG	60	1458
fusA	R	TTACTCGCCGCCGCCGCCAA	60	1458

Allele Sequences

Reference Allele Sequences

Allelic profile of K. variicola 801 (GenBank: CDMV01000001):

leuS (594 bp)

GATCACTGGCCAGACACCGTAAAGACCATGCAGCGCAACTGGATCGGCCGTTCCGAAGGG GTGGAAATCTCCTTTGACGTCAACAACTACGCCGATAAGCTGACCGTCTACACCACCCGT CCGGACACCTTTATGGGCTGCACCTACCTGGCTGTCGCGGCGGGTCACCCGCTGGCGCAG CAGGCTGCAGCGAACAATCCGGCGCTGGCTGCCTTTATCGACGAATGCCGTAATACCAAA GTAGCCGAAGCCGACATGGCGACGATGGAGAAAAAAGGCGTCGATACCGGGTTTAAAGCC ATTCACCCGCTGACCGGCGAAGAAATTCCTGTCTGGGCGGCAAACTTCGTGCTGATGGAA TATGGCACCGGCGCGGTCATGGCCGTTCCGGGTCACGACCAGCGTGACTATGAGTTCGCC AGCAAGTACGGTCTGAACATCAAGCCGGTTATCCTCGCCGCTGACGGCAGCGAGCCGGAT CTCTCCGCGCAGGCGCTGACCGAGAAAGGCGTCCTGTTTAACTCCGGCGAGTTCAGCGGC CTCGATTATGAAGCCGGCTTCAACGCCATTGCCGACAAACTGGCAGCCATGGGC

pgi (600 bp)

GACCGTGCGGTACTGCACGTCGCGCTGCGCAACCGCAGCAATACGCCGATTGTGGTTGAT GGCAAAGATGTGATGCCGGAAGTGAACGCTGTGCTGGAGAAGATGAAAACCTTCTCAGAA GCGATTATCTCCGGTAGCTGGAAAGGCTATACCGGCAAACCGATCACCGACGTGGTTAAC ATTGGTATCGGCGGTTCCGACCTCGGTCCGTTCATGGTCACCGAAGCGCTGCGTCCGTAC AAAAACCACCTCAACATGCACTTTGTCTCCAACGTCGATGGCACCCACATCGCTGAAGTG CTGAAGAACGTCAACCCGGAAACGACCCTGTTCCTGGTGGCGTCCAAGACCTTCACCACT CAGGAAACCATGACCAACGCCCACAGCGCGCGCGACTGGTTCCTGGCGACCGCGGGCGAC GACAAACACGTGGCCAAACACTTCGCGGCGCTTTCTACCAACGCGAAAGCGGTTGGCGAG TTCGGCATCGACACCGCCAACATGTTTGAATTCTGGGACTGGGTTGGCGGCCGTTACTCC CTGTGGTCGGCGATTGGCCTGTCCATCATTCTCTCCGTGGGCTTCGACAACTTCGTTGAG

pgk (444 bp)

GCGTGCGCAGGCCCGCTGCTGGCCGCTGAACTGGACGCGCTGGGTAAAGCGCTGAAAGAG CCGGCTCGTCCGATGGTCGCTATCGTGGGTGGTTCTAAAGTGTCCACCAAACTGACCGTG CTGGATTCCCTGTCTAAAATCGCTGACCAGCTGATCGTCGGCGGCGGTATCGCCAACACC TTCGTTGCTGCCCAGGGCCACAACGTCGGTAAATCCCTGTACGAAGCAGACCTGGTTGAT GAAGCTAAGCGCCTGCTGAGCACCTGTGATATCCCGGTTCCGACGGACGTTCGCGTCGCG ACCGAGTTCTCCGAAACCGCAACGGCAACCCTGAAATCTGTTAACGACATCAAAGATGAC GAGCAGATTCTGGACCTTGGCGACGTTTCCGCACAGAAACTGGCCGACATCCTGAAAAAC GCCAAAACCATCCTGTGGAACGGC

phoE (453 bp)

GATTCCTCTGCCCAGACCGATAACTTTATGACCAAACGCGCCAGCGGCCTGGCGACCTAC CGCAATACCGATTTCTTCGGTCTGGTGGATGGTCTGGATATGACCCTGCAGTACCAGGGT AAAAACGAAGGCCGTGAAGCGAAGAAACAGAACGGCGACGGCGTTGGCACCTCATTAAGC TATGATTTCGGCGGCAGCGACTTCGCCGTCAGCGCGGCCTACACCAGCTCCGACCGTACC AACGATCAGAACCTGCTGGCCCGCGGCCAGGGTTCGAAAGCGGAAGCCTGGGCGACCGGC CTGAAATATGACGCCAACAATATCTACCTGGCGACCATGTATTCTGAGACCCGCAAAATG ACCCCGATCAGCGGCGGTTTTGCCAACAAAGCGCAGAACTTTGAAGCGGTGGCGCAGTAT CAGTTCGACTTCGGTCTGCGTCCGTCCCTCGGC

pyrG (522 bp)

ACGTCCTTGCGCATCGCACTGATTGGCCGTGAGTCAGACCACCGTCTGGTCTACCCCGCC ACGCTTGGCTCACTGGGTGATGCCGCGGCGTCGCTGGGGCTGAACCTGGAGGTCCATTTC TTCGCGCCGGTCAGTCTGTCCCCTGGGTTACACGACCTCCACGACGTCCAGGGCGTCGTG TTGCCAGGCGGTTCCTCGATGGCCGCCGTCAAGGGACAAATCCGCGTGGCGGAAGATACG CTTGCCCGCGGACAGCCCACGCTGGGGTTATGCCTGGGAATGCAGAGCATGATGACCGCA GTCGTGCGCCGACGTCCGGGTTGCGAATCAGCCATTCCCGCCGAAGTGGCTCCGGATGAG GCTCTGCACAGCTTTGTTCCCTTTGTTGATGGCCGTCATCGCTGCGGCGTATTCCCTTTT TCCTTCGGGTTGATCCCCGGTGATATCCGCGAAATGCACTACAACCATCGCTACTGCTTT AACACCGACTTATTACCGGCGCTGGATCGCAGCGGGGTCAGC

rpoB (513 bp)

ATCTCCGCACTCGGCCCAGGCGGTCTGACCCGTGAACGCGCAGGCTTCGAAGTTCGAGAC GTACACCCGACGCACTACGGTCGCGTATGTCCAATCGAAACGCCTGAAGGTCCGAACATC GGTCTGATCAACTCCCTGTCCGTGTACGCACAGACTAACGAATACGGTTTCCTTGAGACC CCGTATCGTAAAGTGACCGACGGTGTGGTTACCGATGAAATTCACTACCTGTCTGCTATC GAAGAAGGCAACTACGTTATTGCTCAGGCGAACTCCAACCTGGATGAAAACGGCCACTTC GTAGAAGACCTGGTTACCTGCCGTAGCAAAGGCGAATCCAGCTTGTTCAGCCGCGACCAG GTTGACTACATGGACGTATCCACCCAGCAGGTGGTATCCGTCGGTGCGTCCCTGATCCCG TTCCTGGAGCACGATGACGCCAACCGTGCATTGATGGGTGCGAACATGCAACGTCAGGCG GTTCCGACTCTGCGCGCTGATAAGCCGCTGGTT

fusA (561 bp)

AACCAGACCATCATCGCCGGTATGGGTGAGCTGCACCTCGACATCATCGTTGACCGTATG AAGCGTGAATTCAACGTTGAAGCGAACGTCGGTAAACCTCAGGTTGCTTACCGTGAAGCG ATTCGCGCGAAAGTTACCGATATCGAAGGTAAACACGCCAAGCAGTCTGGTGGTCGTGGT CAGTACGGTCACGTTGTGATCGACATGTACCCGCTGGAGCCGGGCTCTAACCCGAAAGGT TACGAGTTCATCAACGACATCAAAGGTGGTGTAATTCCTGGCGAATACATCCCGGCCGTT GATAAAGGCATCCAGGAACAGCTGAAAGCTGGTCCGCTGGCAGGTTACCCGGTAGTTGAT ATGGGTATCCGTCTGCACTTCGGTTCTTACCACGACGTTGACTCCTCTGAGCTGGCGTTT AAACTGGCTGCGTCTATTGCCTTTAAAGAAGGCTTTAAGAAGGCAAAACCAGTTCTGCTT GAGCCGATCATGAAGGTTGAAGTAGAAACTCCGGAAGAGAACACTGGTGATGTTATCGGT GACTTGAGCCGTCGTCGCGGT

Sequence Analysis Guidelines

Sequence Type Assignment

This page was designed for assignation of sequence type (ST) for K. variicola isolates, using data for both Sanger and whole genome sequencing technologies.

For Whole Genome Sequencing (WSG)

For WGS we included, first, the ANI (average nucleotide identity; see references below) analysis for proper identification of bacterial species among the Klebsiella pneumoniae species complex (KpSC), using the following genomes as references:

Klebsiella variicola Reference Strain F2R9 (DSM 15968, ATCC BAA-830) (CP072130)
Klebsiella pneumoniae (ATCC 700721, MGH 78578) (CP000647)
Klebsiella quasipneumoniae ATCC 700603 (CP014696)
Klebsiella quasivariicola KPN1705 (CP022823) (this last recently described)
Klebsiella africana SB5857 (GCF_900978845.1)
Klebsiella variicola subs tropica SB94 (GCF_900978435.1)

Only for K. variicola genomes the ST will be determined.

Key References

DNA-DNA hybridization values and their relationship to whole-genome sequence similarities

Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007.

Int J Syst Evol Microbiol 57:81-91. 10.1099/ijs.0.64483-0

Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis

Figueras MJ, Beaz-Hidalgo R, Hossain MJ, Liles MR. 2014.

Genome Announc. 4;2. pii: e00927-14. doi: 10.1128/genomeA.00927-14

Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans

Esperanza Martínez-Romero, Nadia Rodriguez-Medina, Marilu Beltrán-Rojel, Jesús Silva-Sanchez, Humberto Barrios-Camacho, Ernesto Perez-Rueda, Ulises Garza-Ramos.

Salud Publica de Mexico. 2018. 60;1:56-62.