Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry

Abstract

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

Figure 1. Identification of the delta-toxin peak using purified delta-toxin and isogenic strains.

Purified delta-toxin from wild-type Staphylococcus aureus (500 ng) or strains mutated in the accessory gene regulator (agr) locus were spotted on the target and analysed in the Matrix Assisted Laser Desorption Ionization – Time-of-Flight mass spectrometer with a mass-to-charge ratio (m/z) range of 2800 to 3200 Thomson (Th). Setting were as follows: mass range 2000–20000 Th; laser power 80 Volts; pulse extraction 8330 Th; number of laser fires per sample 500; noise cut off 10 mVolts with a minimum resolution of 300; Auto quality mode activated. The settings of the detector were according to the linear mode with a positive source of 20000 Th and a negative pulsed extraction at 2100 Th. The arrow in panel A indicates delta-toxin; additional peaks of 3024, 3040 and 3056 Th correspond to contaminants resulting from the purification of the toxin from S. aureus culture supernatant. Isogenic strains were: RN6390 (delta positive strain) RN6911 (full agr knock-out, delta negative strain) and LUG 950 (rnaIII knock-out, delta negative strain).

Figure 2. Delta-toxin peak in spectra from clinical strains.

Whole-Cell Matrix Assisted Laser Desorption Ionization – Time-of-Flight mass spectrometry analysis of 6 representative clinical strains from the collection of 168. BE1103 3028 and BE1046 1395 are two delta-toxin positive strains showing 3005±5 Thomson (Th) peak; BE1104 4293 and BE1050 5040 are 2 strains expressing a mutated G10S delta-toxin; they exhibited no peak at 3005±5 Th but an additional peak at 3035±5 Th; BE1106 5397 and BE1048 2354 are two delta-toxin negative strains showing no peak at 3005±5 Th or at 3035±5 Th. Settings of the mass spectrometer are the same as in Figure 1.

Figure 3. Northern blot analysis of accessory gene regulator-RNAIII.

Lane 1, positive control isolate (delta-toxin producer); lanes 2 to 18 correspond to the 17 delta-toxin negative clinical isolates. 5S rRNA was used as loading control.