Staphylococcus hominis subspecies can be identified by SDS-PAGE or MALDI-TOF MS profiles

Abstract

Staphylococcus hominis is part of the normal human microbiome. Two subspecies, S. hominis hominis (Shh) and S. hominis novobiosepticus (Shn), have clinical significance. Forty-nine S. hominis isolates were analyzed by the MicroScan automated system, SDS-PAGE and MALDI-TOF methods, followed by partial sequencing of the 16S rDNA gene. The trehalose fermentation test, disk diffusion and broth microdilution tests were used to identify (novobiocin test) and access the susceptibility to oxacillin and vancomycin of isolates. The SCCmec elements and genomic diversity were evaluated by PCR and PFGE methods, respectively. Profiles of 28 (57%; 8 Shh and 20 Shn) isolates corroborated with the results found in all the applied methods of identification. The remaining 21 (43%) isolates were phenotypically identified as Shh by MicroScan; however, they were identified as Shn by SDS-PAGE and mass spectral, and confirmed by 16S rDNA sequencing. Among 41 isolates identified as Shn by the molecular and mass spectrometry methods, 19 (41%) were novobiocin-sensitive, and the trehalose test indicated 11 positive isolates, which are considered atypical phenotypic results for this subspecies. In addition, 92.7% of the isolates identified as Shn by these methods carried mecA gene, while only 12.5% of the Shh isolates were positive. Together, the results highlighted the SDS-PAGE and MALDI-TOF MS methods as promising tools for discriminating S. hominis subspecies.

Figure 1

Dendrogram of the pulsed-field gel electrophoresis (PFGE) profiles of SmaI and ApaI-digested genomic DNA of 49 Staphylococcus hominis spp. isolates and associated characteristics. Similarities percentage is identified on dendrogram derived from the unweighted pair group method using arithmetic averages and based on Dice coefficients; +: positive; −: negative; SCCmec: Staphylococcal chromosomal cassette mec; MIC: minimum inhibitory concentration in µg/mL; oxa: oxacillin; van: vancomycin; novob: novobiocin; na: not-applicable; NT: non-typeable; ^asubspecies identification based on total proteins profile by SDS-PAGE and MALDI-TOF, and by partial sequencing of the 16S rDNA gene; ^bisolates submitted to partial rDNA 16S sequencing; ^cisolates identified as Shh by the Microscan automated system; ^dabsence of ccr complex.

Figure 2

Overview of the mass spectra by MALDI-TOF and proteins profile in SDS-PAGE of S. hominis hominis in comparison to the mass spectrum of and S. hominis novobiosepticus. (A) SDS-PAGE showing the proteins profiles of S. hominis hominis (Shh) in comparison to the S. hominis novobiosepticus (Shn). Arrows (→) indicate the prominent proteins for each subspecies; (B) Distribution of the detected masses by MALDI-TOF mass according to m/z spectra peaks obtained from Shh and Shn. Values in bold means spectra peaks found in Shh or Shn; (E) Section of 350 bp amplicon fragment (base 102 to 151) obtained by DNA sequencing showing the Single Nucleotide Polymorphism (SNP 112:G→A) of rDNA 16S amplicon, which discriminate Shh and Shn. (C,D) *Each peak represents a different mass (m/z) of an intact protein detected in the analyses for Shh or Shn.