The effects of iclaprim on exotoxin production in methicillin-resistant and vancomycin-intermediate Staphylococcus aureus

Abstract

Purpose: Extracellular protein toxins contribute to the pathogenesis of Staphylococcus aureus infections. The present study compared the effects of iclaprim and trimethoprim - two folic acid synthesis inhibitors - with nafcillin and vancomycin on production of Panton-Valentine leukocidin (PVL), alpha haemolysin (AH) and toxic-shock syndrome toxin I (TSST-1) in methicillin-resistant and vancomycin-intermediate S. aureus (MRSA and VISA, respectively).

Methodology: Northern blotting and RT-PCR were used to assess gene transcription; toxin-specific bioassays were used to measure protein toxin production.

Results: As shown previously, sub-inhibitory concentrations (sub-MIC) of nafcillin increased and prolonged MRSA toxin gene transcription and enhanced PVL, TSST-1 and AH production. Sub-inhibitory doses of iclaprim and trimethoprim delayed maximal AH gene (hla) transcription and suppressed AH production; both drugs delayed, but neither reduced, maximal TSST-1 production. Trimethoprim significantly increased lukF-PV expression and PVL production compared to both untreated and iclaprim-treated cultures. Higher concentrations of iclaprim and trimethoprim markedly suppressed MRSA growth, mRNA synthesis and toxin production. In VISA, iclaprim, vancomycin and nafcillin variably increased tst and hla expression, but only nafcillin increased toxin production. Despite its ability to increase hla expression, iclaprim was the most potent inhibitor of AH production.

Conclusions: We conclude that, due to its ability to suppress toxin production, iclaprim should be effective against severe staphylococcal infections caused by toxin-producing MRSA and VISA strains, especially given its ability to concentrate at sites of infection such as skin and skin structures and the lung.

Keywords: Iclaprim; MRSA; Panton–Valentine leukocidin; Staphylococcus aureus; TSST-1; VISA; alpha haemolysin.

Fig. 1.

Effects of antibiotics on MRSA growth at higher inoculum sizes. First-generation toxin assays required a high starting inoculum (>10⁷ c.f.u. ml⁻¹) to ensure detectable levels of toxin were achieved. Because standard MIC determinations are defined using 10⁵ c.f.u. ml⁻¹ and because in vitro efficacies of antibiotics are variably sensitive to increasing inoculum size (i.e. inoculum effect), these studies examined which doses of antibiotic were sub-inhibitory with higher bacterial concentrations. MRSA 1560 amd 04–014 were cultured as described in the text and adjusted to a starting concentration of 1−5×10⁷ c.f.u. ml⁻¹. Iclaprim, trimethoprim and nafcillin (at 0.1, 1.0 or 5× MIC) were added at mid-log phase and samples were taken at various times before, and up to 52 h after, antibiotic addition for bacterial quantitation. Nafcillin at 5× MIC was strongly bactericidal (not shown) and thus was not studied further. Data are from one representative experiment of two.

Fig. 2.

Effects of antibiotics on exotoxin gene expression in MRSA. MRSA were cultured using high initial inoculum conditions as described in Fig. 1. Before, and at various times after, antibiotic addition (0.1, 1.0 and 5× MIC), expression of hla, lukf and tst was analysed by Northern blotting. The lower two bands are total RNA loading controls.

Fig. 3.

Effect of antibiotics on exotoxin protein production in MRSA. Methods used are as described in Fig. 1 except that MRSA were cultured in the presence of only 0.1× MIC (i.e. sub-inhibitory) doses of nafcillin, iclaprim or trimethoprim, or vehicle control. MRSA 1560 (top left, right) produces AH and PVL but not TSST-1; MRSA 04–014 (bottom left, right) produces AH and TSST-1 but not PVL. Samples were removed at various times after antibiotic addition, rendered bacteria-free by centrifugation and sterile filtration, concentrated by centrifugal 10 000 mwt filter, then aliquoted and frozen until assayed for toxins of interest as described in the text. Data are given as the means of duplicate measurements±standard deviation from one experiment.

Fig. 4.

Comparison of growth dynamics and toxin production in MRSA and VISA under low inoculum conditions. MRSA strain 04–014 and VISA strain Mu50 have similar exotoxin profiles (i.e. hla and tst positive; pvl negative). (a) Using low initial inoculum conditions (cultures started with ~1×10⁵ c.f.u. ml⁻¹), growth in untreated cultures was followed over time by optical density at 600 nm. (b) Relative tst expression and (c) production of TSST-1 protein in untreated cultures were measured at 10 h and 24 h by qRT-PCR and ELISA, respectively. (d) Expression of hla and (e) production of alpha haemolysin were measured by qRT-PCR and rabbit erythrocyte lysis assay, respectively, in two untreated strains of MRSA and in the VISA Mu50 strain. *=below the level of detection. Data shown are the means of duplicate measurements±standard deviation from a single experiment.

Fig. 5.

Effects of antibiotics on toxin gene expression and protein production in VISA. Cultures were initiated with ~1×10⁵ c.f.u. ml⁻¹ and antibiotics were added such that the final concentrations were 1/4th, 1/8th or 1/16th the MIC. The effects of antibiotics on expression of tst and hla at 10 h (a and c, respectively) or on protein production at 24 h (b and d, respectively) were measured as described above. ICL: iclaprim; TMP: trimethoprim; VAN: vancomycin; NAF: nafcillin. Data shown are the means of triplicate measurements on each samples obtained from a single experiment.