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Background   Klebsiella  spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new  Klebsiella  species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore  Klebsiella  group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence.    Methods  We analysed whole-genome sequence data of a diverse selection of  Klebsiella  spp. isolates and identified resistance and virulence factors. Using the genomes of 3594  Klebsiella  isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from  Klebsiella  species were performed using generalized linear mixed-effects models.    Results  Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight  Klebsiella  species can be distinguished using MALDI-TOF MS. We identified  K. pneumoniae  (71.2%;  n  = 12,523),  K. quasipneumoniae  (3.3%;  n  = 575),  K. variicola  (9.8%;  n  = 1717), “ K. quasivariicola ” (0.3%;  n  = 52),  K. oxytoca  (8.2%;  n  = 1445),  K. michiganensis  (4.8%;  n  = 836),  K. grimontii  (2.4%;  n  = 425) and  K. huaxensis  (0.1%;  n  = 12). Isolates belonging to the  K. oxytoca  group, which includes the species  K. oxytoca ,  K. michiganensis  and  K. grimontii , were less often resistant to 4th-generation cephalosporins than isolates of the  K. pneumoniae  group, which includes the species  K. pneumoniae ,  K. quasipneumoniae ,  K. variicola  and “ K. quasivariicola ” (odds ratio = 0.17,  p  < 0.001, 95% confidence interval [0.09,0.28]). Within the  K. pneumoniae  group, isolates identified as  K. pneumoniae  were more often resistant to 4th-generation cephalosporins than  K. variicola  isolates (odds ratio = 2.61,  p  = 0.003, 95% confidence interval [1.38,5.06]) . K. oxytoca  group isolates were found to be more likely associated with invasive infection to primary sterile sites than  K. pneumoniae  group isolates (odds ratio = 2.39,  p  = 0.0044, 95% confidence interval [1.05,5.53]).    Conclusions  Currently misdiagnosed  Klebsiella  spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS.  Klebsiella  groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13073-021-00960-5.

genome medicine

Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study