Culture conditions and sample preparation methods affect spectrum quality and reproducibility during profiling of Staphylococcus aureus with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

UNLABELLED: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a promising tool to rapidly characterize Staphylococcus aureus. Different protocols have been employed, but effects of experimental factors, such as culture condition and sample preparation, on spectrum quality and reproducibility have not been rigorously examined. We applied MALDI-TOF MS to characterize a model system consisting of five methicillin-sensitive (MSSA) and five methicillin-resistant S. aureus isolates (MRSA) under two culture conditions (agar and broth) and using two sample preparation methods [intact cell method and protein extraction method (PEM)]. The effects of these treatments on spectrum quality and reproducibility were quantified. PEM facilitated increases in the number of peaks and mass range width. Broth cultures further improved spectrum quality in terms of increasing the number of peaks. In addition, PEM increased reproducibility in samples prepared using identical culture conditions. MALDI imaging data suggested that the improvement in reproducibility may result from a more homogeneous distribution of sample associated with the broth/PEM treatment. Broth/PEM treatment also yielded the highest rate (96%) of correct classification for MRSA. Taken together, these results suggest that broth/PEM maximizes the performance of MALDI-TOF MS to characterize S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Two culture conditions (agar or broth) and two sample preparation methods (intact cell or protein extraction) were evaluated for their effects on profiling of Staphylococcus aureus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results indicated that MALDI-enabled profiling of S. aureus is most effective when cultures are grown in broth and processed using a protein extraction-based approach. These findings should enhance future efforts to maximize the performance of this approach to characterize strains of S. aureus.

Staphylococcal exfoliative toxins cleave alpha- and beta-melanocyte-stimulating hormones.

The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains of Staphylococcus aureus and are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide beta-melanocyte-stimulating hormone (beta-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving alpha-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving beta-MSH.

Mutational analysis of the superantigen staphylococcal exfoliative toxin A (ETA).

Exfoliative toxin A (ETA) is known to be a causative agent of staphylococcal scalded skin syndrome (SSSS). Although relatively little is known about exactly how the exfoliative toxins (ETs) cause SSSS, much has been discovered recently that may help elucidate the mechanism(s) by which ETA exhibits activities such as lymphocyte mitogenicity and epidermolytic activity. Here, we have shown that highly purified ETA does have T lymphocyte mitogenic activity in that wild-type ETA induced T cell proliferation whereas several single amino acid mutants lacked significant activity. Neither wild-type ETA nor any single amino acid mutants were proteolytic for a casein substrate, yet esterase activity was detected in wild-type ETA and several mutants, but eliminated in other mutants. A mutation in aa 164 (Asp to Ala) showed a 9-fold increase in esterase activity as well. Finally, we correlated esterase activity with epidermolytic activity. All mutants that lost esterase activity also lost epidermolytic activity. Conversely, mutants that retained esterase activity also retained exfoliative activity, implicating serine protease or serine protease-like activity in the causation of SSSS. Moreover, the mutants that displayed markedly reduced T cell superantigenic activity retained their epidermolytic activity (although some of these mutants required higher doses of toxin to cause disease), which suggests an ancillary role for this activity in SSSS causation.

Unique superantigen activity of staphylococcal exfoliative toxins.

Certain strains of Staphylococcus aureus express one or both of two related, but immunologically distinct, exfoliative toxins (ETA and ETB). These toxins induce the symptoms associated with staphylococcal scalded skin syndrome. Both ETs have been shown to stimulate T cell proliferation. Recently, it was reported that ETA is a superantigen that stimulates T cells bearing human Vbeta2 or several murine Vbetas. However, other investigators have proposed that the superantigenicity reported for ETA resulted from contaminants in commercial preparations. This present study addresses those conflicting reports by assessing the biological and immunologic activities of highly purified rETs. ETA and ETB required APCs to induce selective polyclonal expansion of several human Vbetas (huVbetas), although, neither toxin expanded huVbeta2. ETB induced expansion of murine T cells bearing Vbetas 7 and 8, those that have the highest homology to the huVbetas expanded by ETA and ETB. Although flow cytometry of ETB-stimulated T cells matched PCR results, stimulation by ETA reduced percentages of T cells positive for several huVbetas that had been shown to have increased levels of mRNA transcripts. ETA and ETB induced contrasting reactions in vivo. In rabbits, ETB was moderately pyrogenic and enhanced susceptibility to lethal shock, while ETA lacked both activities. Predictions based on comparisons with other superantigens suggest molecular regions potentially involved in receptor binding in the ETA crystal structure and a modeled ETB three-dimensional structure. These results show that ETs are superantigens with unique properties that could account for the discrepancies reported.

Molecular epidemiology of staphylococcal scalded skin syndrome in premature infants.

BACKGROUND: Outbreaks of nosocomial staphylococcal scalded skin syndrome (SSSS) in infants have been well-described associated with the well baby nursery or delivery room. We describe two cases of SSSS in very low birth weight infants in a neonatal intensive care unit (NICU) and the success of infection control strategies used to prevent an outbreak. METHODS: Staphylococcal scalded skin syndrome was diagnosed in two infants in the NICU: Case I (a 47-day-old, formerly 530-g female); and Case II diagnosed 48 h later (a 41-day old, formerly 706-g female). Multiple infection control measures were implemented: (1) isolation and intravenous antibiotic treatment of cases; (2) placement of exposed infants into a cohort; (3) prophylactic mupirocin treatment of the anterior nares of all infants in the NICU and staff colonized with Staphylococcus aureus; and (4) personnel hand washing with hexachlorophene. Detection of exfoliative toxin A and studies to determine the genetic relatedness of S. aureus strains isolated from patients and staff were performed. RESULTS: In addition to the two SSSS cases, S. aureus was isolated from 2 of 12 (17%) exposed asymptomatic infants, 2 of 20 (10%) ancillary staff, 8 of 30 (27%) nurses and 6 of 24 (25%) physicians. Exfoliative toxin A-producing strains were isolated from both cases and one asymptomatic infant. No toxin was expressed by strains isolated from staff. Pulse field gel electrophoresis demonstrated genetically identical strains of S. aureus from the two SSSS cases and the asymptomatic infant, whereas three staff members harbored strains genetically related to the case strain. Unexpectedly two additional unique clusters of genetically related S. aureus strains were identified from the surveillance cultures. CONCLUSIONS: This report documents the rare occurrence of nosocomial SSSS attributed to transmission in the NICU among extremely low birth weight infants. Multiple infection control strategies were effective in limiting the outbreak. Molecular epidemiology investigation supported a unique S. aureus strain responsible for this event and the presence of bidirectional spread between staff and patients of non-toxin-producing strains.

The structure of the superantigen exfoliative toxin A suggests a novel regulation as a serine protease.

Exfoliative toxin A (ETA) causes staphylococcal scalded skin syndrome which is characterized by a specific intraepidermal separation of layers of the skin. The mechanism by which ETA causes skin separation is unknown although protease or superantigen activity has been implicated. The X-ray crystal structure of ETA has been solved in two crystal forms to 2.1 and 2.3 A resolution and R-factors of 17% and 19%, respectively. The structures indicate that ETA belongs to the chymotrypsin-like family of serine proteases and cleaves substrates after acidic residues. The conformation of a loop adjacent to the catalytic site is suggested to be key in regulating the proteolytic activity of ETA through controlling whether the main chain carbonyl group of Pro192 occupies the oxyanion hole. A unique amino-terminal domain containing a 15-residue amphipathic alpha helix may also be involved in protease activation through binding a specific receptor. Substitution of the active site serine residue with cysteine abolishes the ability of ETA to produce the characteristic separation of epidermal layers but not its ability to induce T cell proliferation.