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Effective surveillance and management of pathogenic  Escherichia coli  relies on robust and reproducible typing methods such as multilocus sequence typing (MLST). Typing of  E .  coli  by MLST enables tracking of pathogenic clones that are known to carry virulence factors or spread resistance, such as the globally-prevalent ST131 lineage. Standard MLST for  E .  coli  requires sequencing of seven alleles, or a whole genome, and can take several days. Here, we have developed and validated a nucleic-acid-based MALDI-TOF mass spectrometry (MS) method for MLST as a rapid alternative to sequencing that requires minimal operator expertise. Identification of alleles was 99.6% concordant with sequencing. We employed MLST by MALDI-TOF MS to investigate diversity among 62  E .  coli  isolates from Sydney, Australia, carrying a  bla   CMY-2 -like gene on an IncI1 plasmid to determine whether any dominant clonal lineages are associated with the spread of this globally-disseminated resistance gene. Thirty-four known sequence types were identified, including lineages associated with human disease, animal and environmental sources. This suggests that the dissemination of  bla   CMY-2 -like-genes is more complex than the simple spread of successful pathogenic clones.  E .  coli  MLST by MALDI-TOF MS, employed here for the first time, can be utilised as an automated tool for large-scale population analyses or for targeted screening for known high-risk clones in a diagnostic setting.

MALDI-TOF Mass Spectrometry for Multilocus Sequence Typing of Escherichia coli Reveals Diversity among Isolates Carrying blaCMY-2-Like Genes