Protocols used for MLST of Moraxella catarrhalis

Genes

The M. catarrhalis MLST scheme uses internal fragments of the following eight house-keeping genes:

• glyRS (glycyl-tRNA synthetase beta subunit)

• ppa (Pyrophosphate phospho-hydrolase)

• efp (elongation factor P)

• fumC (fumarate hydratase)

• trpE (anthranilate synthase component I)

• mutY (adenine glycosylase)

• adk (Adenylate kinase)

• abcZ (ATP-binding protein)

PCR Amplification

The primer pairs we use for the PCR amplification of internal fragments of these genes are:

		Product length:
glyRS:	F 5’-GCACCGAAGAGTTGCCACCA-3’	762 bp
glyRS:	R 5’-ACGCAACGGGCAAATCCACC-3’
ppa:	F 5’-AATAAAATTCTAGATGCTGGC-3’	523 bp
ppa:	R 5’-ACTTATTGCTCTGTCCAGCG-3’
efp:	F 5’-CTCTGATTGACAACTGGCAGG-3’	582 bp
efp:	R 5’-GATATTCGCCAGTACGCG-3’
fumC:	F 5’-GGGCGGTACAGCAGTCGGCAC-3’	675 bp
fumC:	R 5’-CTCATCAAATTCAGCTTCAG-3’
trpE:	F 5’-TTATCCCGCATCGAAAATGG-3’	545 bp
trpE:	R 5’-GGTTTCATCCCATTCAGCC-3’
mutY:	F 5’-GGCAATACCATCATCAGCCG-3’	609 bp
mutY:	R 5’-GGTAACTGACTTTGAACGCC-3’
adk:	F 5’-GGCATTCCTCAAATCTCAAC-3’	631 bp
adk:	R 5’-GATGGGCTTTATTGTCAAATG-3’
abcZ:	F 5’-ACATGCTGATGATGGTGAG-3’	610 bp
abcZ:	R 5’-CACTGGCAAGTTCAAGCGC-3’

An annealing temperature of 52° C is fine for all genes, except glyRS (58° C) and adk (54° C).

Sequencing

We use the amplification primer pairs for the sequencing step except for the following genes.

glyRS: F 5’-GCACCGAAGAGTTGCCACCA-3’ glyRS: R 5’-ATATCGGCTTGACGCTGATC-3’

fumC: F 5’-GCTGTCAAAGTCGCTAAAG-3’ fumC: R 5’-CTCATCAAATTCAGCTTCAG-

mutY: F 5’-TATGCTGTGTGGGTATCTG-3’ mutY: R 5’-GGTAACTGACTTTGAACGCC-3’

Allele template

Allelic profile of M. catarrhalis MC216

glyRS (537 bp):

GTTGGCACTTTACTAAATGATCAAAATATCAGCTTTGATGATATTCATGCCTTTGCCGCACCACGCCGTTTGGCACTTACTATTAC
AGGTGTGGGCGAGTTTCAGCCTGACCGTATCGAAACTAAAAAAGGCCCAAGTGTTAAGGCAGCGTTTGATGACGCAGGTAATTTAA
CGCGTGCAGGGCAAGGATTTTTGCAAGGTTTAAATGCCACTGGGCGAAATCTTAGTGTCAAAGATTTGGGGCGTATTGCTGATAAA
AAAGGCGAGTACATCGCTTATGATTTGACCATTAAAGGCGAAAAAATCACCGATTTAATGCCAAATATCTTACAAAAAGCGCTTGA
TGATTTGCCAATTGCTAAGCGTATGCGTTCTGGCATTGATCGCCATGAGTTCATACGGCCTGTTCATTGGGTGGTTTTGATGTCAG
ATGATGTTGTAATTGAGGCGACCATTCAAGGCCATAAAACTGGCAACCAAAGCCGTGGTCATCGCTATCATTCACCTGATTTTTTT
GCCATTGATCATGCCGATCAT

ppa (393 bp):

TCAAATCATAAAATTGAGTGGAATCGTCATTTGGCATGCTTTGAGCTTGATCGTGTTGAGCCAATTGCCTTTGCCAAACCATGCAA
CTATGGCTTTATTCCCCAAACCTTAGATGAAGATGGTGATGAATTAGACGCCTTAATCATCACAGAACAGCCTTTGACGACGGGTA
TTTTTCTTAAAGCCAAAGTCATCGGGGTAATGAAATTTGTGGATGATGGCGAGGTCGACGATAAAGTCATCGTTGTTCCTGCCGAT
GATCGTAATAATGGCAACGCCTATAATAGTCTTGACGACCTACCACCACAGTTAATAAAGCAACTTGAATTTCATTTTAGTCATTA
TAAAGACTTAAAAAAACCAGGCTCAACTGTGGTTGAGGGCTTTTTTGAT

efp (414 bp):

AATGAATATGTGAAACCAGGTAAAGGTCAAGCTTTTAACCGTGTTAAGTTGCGTAATCTACGCACAGGCAAAGTGCTTGAGCAGAC
TTTTAAGTCAGGCGATTCACTTGAAGGTGCTGATGTGGTGGATGTGGAAATGAACTATCTATACAACGATGGCGAATTTTGGCATT
TTATGCACCCTGAGACTTTTGAGCAATTGCAAGCAGATAAAACCGCAATGGGTGATGCTGCACAGTGGCTAAAAGAAAATTCAAAT
GCGTTATGCACTTTGACGCTATTTAATGGTGCGCCGCTGTCGGTCACACCACCAAACTTTGTAGAGCTTGAAATCGTTGAAACTGA
CCCAGGTGTTCGCGGTGATACCTCAGGTGGTGGCGGTAAGCCTGCACGCCTTGAAACAGGTGCAACCGTG

fumC (465 bp):

GAGGCTTTGGCCGCACGAGATGCCGAAGTATTTGCTTCAGGTGCACTAAAAACACTGGCAGCAAGCCTAAATAAAATCGCCAATGA
TGTGCGTTGGCTGGCTTCAGGCCCACGCTGCGGTCTAGGTGAGCTTAGCATTCCTGAAAATGAGCCTGGTTCATCCATCATGCCTG
GTAAAGTCAATCCAACTCAGTGTGAGGCGATGACCATGGTGGTCACCCAAGTCTTTGGTAATGATACGACCATTTCTATGGCAGGG
GCTTCTGGTAATTTTGAATTAAATGTTTATATGCCTGTGATTGGATATAACTTATTGCAGTCGATTCGTCTGCTAACGGATGCGAT
CAATAGCTTTAATGAGCATTGTGCGGTAGGGATCGAACCAATTTACGAAAAAATTGACCATTATTTACATCATTCTTTGATGTTGG
TCACCGCACTCAATCGCCATATTGGCTATGAAAAT

trpE (372 bp):

TTAAATGACCAAAAAGAGATCGCAGAACATCTGATGCTCATTGATCTTGGGCGTAATGATATTGGGCGTGTCTGTGAGATTGGTTC
GGTTAAAGTGACAGATCAGATGTTCATTGAACGCTACTCGCAAGTGATGCATATCGCTAGTAATGTTGAGGGGCGTATACGCCAAG
ATGTCGATGCTTTAGATGTGTTTTGTGCGACCTTTCCAGCGGGTACTTTGTCTGGTGCGCCAAAAATCCGTGCCATGCAAATTATT
GATGAAGTTGAGCCAGTGCGTCGCACCGTTTTTGGCGGGGCGATCGGATATCTTGGCTGGCATGGCAATATGGATACTGCCATCGC
AATACGCACAGCGGTCATGAAAGATGGT

mutY (426 bp):

TTTTTTGAGCCGTTTTTGGCACGATTTGCAACCGTACAAGAGCTTGCTGTAGCAGATTGGCAAGAAGTAGCGTCTTTTTGGGCAGG
GCTTGGGTATTATGCTCGGGCAAGAAATTTACACGCAGGCGCACAGCAAGTGGCTGATTTTATTGATACTCATGGCAGGTTTCCTG
AAACGGTCAATGAGTGGCAAGCAGTCAAAGGGGTGGGGCGATCGACAGCTGGCGCAATCGTTGCGATGGGCGTTAAGAAGTTTGGC
GTCATCTGCGATGGCAATGTCAAGCGTGTACTTGCTCGCCATCGCGCGGTTTTTGGTGATGTGACAAAAAGCGCAACTGACAAAAG
GCTTTGGGAAATTGCCACCGCACTAACCCCGAAAGAGTATAGTGGACACTATGCCCAAGCGATGATGGATTTGGGTGCAACA

adk (471 bp):

CAAGCAAAAAGTGTCATGGATGCAGGTCAATTGGTTTCTGATGAGCTGATTATTAATTTGGTTAAAGAACGCTTAAAACAGCCAGA
TTGTGACAATGGTTGTATTTTTGATGGATTCCCACGCACAATCCCACAAGCCAAAGCACTGGCAGATGCTGGCATTGCCATTGACC
ATGTGATTGAAATCAGCGCCCCTGATGATGAAATCGTAAGCCGTATGTCGGGTCGCCGTGCACATTTGGCATCAGGTCGTACTTAT
CATGTGATCTATAATCCCCCAAAAACTGACGGTATTGATGATGTCACAGGCGAGGCACTGGTGCAGCGTGATGATGATAAAGAAGA
AGTGGTGCGTGATCGCTTATCGGTATATCACGCCCAAACCGCTACATTGATTAGTCATTATCAAGAAGTTGCCAAATCGGGCGAAA
ATGCCCCTGAATATCATGACTTTGATGGAACAAAATCCATC

abcZ (429 bp):

CTTGATGTGGTGATGGAGGGTGATGACAAAGTTGCCCAAGCCTTAAAAAACTATCATCAAGCCAGTGCAAAATGCGCCATGGGCGA
CAATGAAGCTTGTGATGCCATGAGCGGTCTTCAGCAAACACTCGATGAGTTAAATGGTTGGGACTTGGAGCGGCGTGCGCGTGCAT
TGATTGACAGCATGGGGTTTGACGCTGATGCTTGCTTATCAACATTATCAGGGGGTCGTAAACGCCGTGTTCTGTTAGCACAGGCT
TTGGTAACAAATCCTGATGTGCTACTTTTAGACGAGCCAACCAACCATTTAGATATTGAAAGCATCACTTGGCTTGAAAAATTTTT
ATTAAATGAACGATTAACCGTTCTTTTCATCTCACATGATCGGAAGTTTGTTGATACACTTGCCACTTATATCATCGAGCTTGAC