MALDI-TOF MS distinctly differentiates nontypable Haemophilus influenzae from Haemophilus haemolyticus

PLoS One. 2013;8(2):e56139. doi: 10.1371/journal.pone.0056139. Epub 2013 Feb 14.


Nontypable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus exhibit different pathogenicities, but to date, there remains no definitive and reliable strategy for differentiating these strains. In this study, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a potential method for differentiating NTHi and H. haemolyticus. The phylogenetic analysis of concatenated 16S rRNA and recombinase A (recA) gene sequences, outer membrane protein P6 gene sequencing and single-gene PCR were used as reference methods. The original reference database (ORD, provided with the Biotyper software) and new reference database (NRD, extended with Chinese strains) were compared for the evaluation of MALDI-TOF MS. Through a search of the ORD, 76.9% of the NTHi (40/52) and none of the H. haemolyticus (0/20) strains were identified at the species level. However, all NTHi and H. haemolyticus strains used for identification were accurately recognized at the species level when searching the NRD. From the dendrogram clustering of the main spectra projections, the Chinese and foreign H. influenzae reference strains were categorized into two distinct groups, and H. influenzae and H. haemolyticus were also separated into two categories. Compared to the existing methods, MALDI-TOF MS has the advantage of integrating high throughput, accuracy and speed. In conclusion, MALDI-TOF MS is an excellent method for differentiating NTHi and H. haemolyticus. This method can be recommended for use in appropriately equipped laboratories.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriological Techniques / methods
  • Cluster Analysis
  • DNA, Bacterial / genetics
  • Haemophilus / chemistry
  • Haemophilus / genetics
  • Haemophilus / isolation & purification*
  • Haemophilus Infections / diagnosis*
  • Haemophilus Infections / microbiology*
  • Haemophilus influenzae / chemistry
  • Haemophilus influenzae / genetics
  • Haemophilus influenzae / isolation & purification*
  • Humans
  • Phylogeny
  • RNA, Ribosomal, 16S / genetics
  • Reference Standards
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*


  • DNA, Bacterial
  • RNA, Ribosomal, 16S

Grant support

This research was supported by the Acute Meningitis-Encephalitis Syndrome Surveillance project and grants (2011CB504904) from the National Basic Research Program of China (973 Program), the National Key Program for Infectious Disease of China (Contract No. 2008ZX10004-002) and the China Mega-Project for Infectious Disease (Contract No. 2011ZX10004-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.