RESUMO
Previous studies showed that levels as low as 0.1% sodium ultraphosphate (UP), 0.1% sodium polyphosphate glassy (SPG) and 0.5% tetrasodium pyrophosphate (TSPP) were bactericidal and bacteriolytic to early-exponential phase cells of Staphylococcus aureus ISP40 8325. In the present study, Ca2+ (0.01 M) or Mg2+ (0.01 M) reversed the bacteriolytic effects of UP (0.1%) and SPG (0.1%) to S. aureus . In addition, Ca2+ (0.01 M) or Mg2+ (0.01 M), when added to the culture medium before inoculation, protected cells from growth inhibition by UP and SPG. Moreover, the bactericidal effects of UP or SPG were reversed by Ca2+ or Mg2+ in metal-rescue experiments in which the metals were added to polyphosphate-containing medium after 1 h of incubation. No additive effect existed between Mg2+ and Ca2+. Growth inhibition of TSPP was not reversed by Mg2+ or Ca2+, but it was reversed by Fe3+ when Fe3+ was added to protect cells 1 h before the addition of TSPP. These studies show that the antibacterial effects of phosphates can be altered substantially by the metal-ion content of the environment.
RESUMO
Phosphates have been approved for use in meat products primarily to protect flavor and increase yields. It also is known that phosphates have antimicrobial properties. The objective of this study was to compare the effects of different phosphates in a model system. Minimum inhibitory concentrations (MICs) of selected food-grade phosphates added to early-exponential-phase cells of Staphylococcus aureus ISP40 8325 in a synthetic medium were determined to be 0.1% for sodium ultraphosphate and sodium polyphosphate glassy and 0.5% for sodium acid pyrophosphate, sodium tripolyphosphate and tetrasodium pyrophosphate. Thus, the MIC values for the very long chain-length phosphates were lower than the MIC values for shorter chain-length phosphates. Leakage of intracellular nucleotides was observed both spectrophotometrically (release of A260-absorbing material) and microscopically (appearance of gelatinous cellular aggregates). Treatment of the gelatinous cellular aggregates with DNase, RNase and proteinase indicated that the aggregates contained DNA, RNA and protein, thus indicating cellular lysis in the presence of phosphates.
RESUMO
The results of previous studies indicated that the antibacterial effects of long-chain polyphosphates (sodium polyphosphate glassy [SPG] and sodium ultraphosphate [UP]) to Staphylococcus aureus ISP40 8325 could be attributed to damage to the cell envelope (cell wall or cell membrane). Also, Ca2+ (0.01 M) or Mg2+ (0.01 M) reversed the bactericidal and bacteriolytic effects of polyphosphates in S. aureus . In the present study, 0.4 M sodium chloride (NaCl) protected the cells from leakage caused by SPG and 0.6 M NaCl protected the cells from leakage by UP. Polymyxin, a peptide antibiotic that causes cell membrane damage, induced leakage even in the presence of 0.6 M NaCl. In the presence of 0.4 M NaCl, bacterial leakage was significantly reduced by disodium ethylenediamine tetraacetate (EDTA), a metal chelator that causes cell wall damage. Bacterial leakage by polyphosphates was significantly greater at pH 8 than at pH 6, which suggested that metal-ion chelation was involved in the antibacterial mechanism. A dialysis membrane (MWCO 100) was used to separate free metal and polyphosphate-bound metal. Levels of free Ca2+ and Mg2+ in polyphosphate-treated cells were significantly lower than those of the cells without polyphosphate. This free-metal dialysis study provided Chemical evidence to show that long-chain polyphosphates interacted with S. aureus cell walls by a metal-ion chelation mechanism. In addition, long-chain polyphosphates were shown to bind to the cell wall, chelate metals, and remain bound without releasing metal ions from the cell wall into the suspending medium. A hypothesis is proposed in which the antibacterial mechanism of long-chain polyphosphates is caused by binding of long-chain polyphosphates to the cell wall of early-exponential phase cells of S. aureus ISP40 8325. The polyphosphates chelate structurally essential metals (Ca2+ and Mg2+) of the cell wall, resulting in bactericidal and bacteriolytic effects. The structurally essential metals probably form cross bridges between the teichoic acid chains in the cell walls of gram-positive bacteria.