Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea.

The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.

Antimicrobial performance of alkaline ionic fluid (GC-100X) and its ability to remove Escherichia coli O157:H7 from the surface of tomatoes.

An efficacy test of GC-100X, a noncorrosive alkaline ionic fluid (pH 12) composed of free radicals and supplemented with xylitol, was carried out against six major foodborne pathogens-Staphylococcus aureus FRI 913, Salmonella enterica serovar Enteritidis ATCC 13076, S. enterica serovar Typhimurium DT104 Korean isolate, Vibrio parahaemolyticus ATCC 17803, Escherichia coli O157:H7 ATCC 43894, and Pseudomonas aeruginosa KCTC 1637-at three different temperatures (4, 25, and 36 degrees C) with or without organic load (2% yeast extract). Results revealed a more than 4-log10 (CFU/ml) reduction (1.0 x 10(4) CFU/ml reduction) against all pathogens reacted at 37 degrees C for 3 h in the absence of organic material. GC-100X solution diluted with an equal volume of distilled or standard hard water (300 ppm CaCO3) showed effective bactericidal activity, particularly against gram-negative bacteria. Washing efficacy of GC-100X solution was compared against E. coli O157:H7 on cherry tomato surfaces with those of a commercially used detergent and chlorine water (100 ppm). Viable cell counts of E. coli O157:H7 that had penetrated to the cores of tomatoes after sanitizing treatment revealed that GC-100X stock and its 5% diluted solutions had similar washing effects to 100-ppm chlorine water and were more effective than the other kitchen detergent. These results indicate that GC-100X has good bactericidal and sanitizing activities and is useful as a new sanitizer for food safety and kitchen hygiene.

Multidrug-resistant Salmonella typhimurium and Salmonella enteritidis identified by multiplex PCR from animals.

Antibiotic resistance in Salmonella enteritidis and S. typhimurium, one of most frequent etiologic pathogens of food-borne bacterial gastroenteritidis in humans, is a serious health problem worldwide. Fifteen and 22 each of S. enteritidis and S. typhimurium were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns and phage types. S. enteritides isolates were highly resistant to sulfonamides (86.7%) and four of them (26.6%) showed multiple antibiotic resistance. The most frequent phage type (PT) of S. enteritids was PT1 (33.3%) even though none of them had multiple antibiotic resistance. S. typhimurium isolates were highly resistant to streptomycin, sulfonamides, and tetracycline, 100%, 95.5%, and 86.4% respectively. The incidence of multiple antibiotic resistance of S. typhimurium isolates was extremely high (100%) comparing to S. enteritidis isolates (26.7%). Two of the five ACSSuT type S. typhimurium isolates, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, were phage type DT104. All S. typhimurium isolates were sensitive to florfenicol. For the rapid detection of multiple antibiotic resistant S. enteritidis and S. typhimurium isolates, particularly ACSSuT type S. typhimurium DT104, antibiotic resistance genes, cmlA/tetR, PSE-1, and TEM, and Salmonella spp. Specific gene, SipB/C, were amplified using four pairs of primers in hot-started multiplex polymerase chain reaction. Two Korean isolates of S. typhimurium DT104 showed TEM amplicons instead of PSE-1 for the ampicillin resistance. The multiplex PCR used in this study was useful in rapid detection of ACSSuT type S. typhimurium and identification of b-lactamase gene distribution among Salmonella isolates.

Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes.

Zinc overload may be a key mechanism of neuronal death in acute brain injury. We have demonstrated previously that zinc overload neurotoxicity involves protein kinase C (PKC)-dependent rises in intracellular levels of reactive oxygen species (ROS). However, the cascade linking PKC activation to ROS generation in cultured cortical neurons has been unknown. A recent study has demonstrated that ROS-generating NADPH oxidase is present in sympathetic neurons and contributes to NGF deprivation-induced cell death. Because NADPH oxidase is activated by PKC, in the present study, we examined the possibility that NADPH oxidase is the effector for oxidative stress in zinc-overloaded cortical cells. Reverse transcription-PCR and Western blot analyses revealed that naive cultured cortical cells express subunits of NADPH oxidase at low levels. Exposure to zinc substantially increased levels of NADPH oxidase subunits in both neurons and astrocytes. In addition, zinc exposure induced translocation of the p47(PHOX) and p67(PHOX) subunits to the membrane, a signature event for NADPH oxidase activation. Addition of a selective PKC inhibitor, GF109203X, blocked both the induction and the membrane translocation of NADPH oxidase by zinc. Supporting the role for NADPH oxidase in zinc-triggered oxidative injury, NADPH oxidase inhibitors attenuated ROS production and cortical neuronal death induced by zinc. In addition, Cu/Zn-superoxide dismutase and catalase attenuated zinc-induced cortical neuronal death. Our results have demonstrated that zinc overload induces and activates NADPH oxidase in cortical neurons and astrocytes in a PKC-dependent manner. Thus, NADPH oxidase may be an enzyme contributing to ROS generation in zinc-overloaded cortical neurons and astrocytes.

A novel neuroprotective mechanism of riluzole: direct inhibition of protein kinase C.

In addition to its antiexcitotoxic action, the anti-amyotrophic lateral sclerosis (ALS) neuroprotectant riluzole protects against nonexcitotoxic oxidative neuronal injury. In light of evidence that protein kinase C (PKC) mediates oxidative stress in cortical culture, we examined the possibility that riluzole's antioxidative neuroprotection involves PKC inhibition. Riluzole (30 microM) blocked phorbol 12-myristate 13-acetate (PMA)-induced increases in membrane PKC activity in cultured cortical cells. Suggesting a direct action, riluzole also inhibited the activity of purified PKC. Consistently, both PKC depletion and oxidative neuronal death induced by PMA were markedly attenuated by riluzole. The site of action of riluzole on PKC was not likely the diacylglycerol binding site but the catalytic domain, since riluzole did not alter radiolabeled phorbol-12,13-dibutyrate binding, but inhibited PKM, the catalytic domain of PKC. However, increasing ATP concentrations did not alter the inhibition of PKC by riluzole, making it unlikely that riluzole is a competitive inhibitor of ATP binding at PKM. Present results have demonstrated that riluzole directly inhibits PKC, which action may contribute to its antioxidative neuroprotective effects. In addition, it appears possible that PKC inhibition may be able to explain some of its well-known channel inhibitory and neuroprotective effects. Combined with findings that PKC activity is increased in ALS, the present results suggest that PKC may be a potential therapeutic target in ALS.

Mediation by membrane protein kinase C of zinc-induced oxidative neuronal injury in mouse cortical cultures.

Transsynaptic movement of endogenous zinc may play a key role in selective neuronal death after brain ischemia and prolonged seizures. As to the mechanism, we have reported recently that zinc-induced neuronal death occurs mainly by oxidative stress in cortical cultures. Here we present evidence supporting the idea that activation of membrane protein kinase C (PKC) in neurons is likely to play a key role in zinc-induced oxidative neuronal injury. Exposure of cortical cultures to 300 microM zinc for 15 min induced increases in the activity, without changing the amount, of membrane PKC to two- to threefold of control values, followed by neuronal death over the next day. Addition of a zinc chelator, Ca-EDTA, or PKC inhibitors with zinc completely abolished the zinc-induced increase in the membrane PKC activity. Indicating the participation of PKC in zinc-induced oxidative stress and neuronal death, the selective PKC inhibitor GF109203X attenuated both. Furthermore, as in zinc-induced neuronal death, activation of PKC with phorbol esters induced free radical generation and neuronal death, which were blocked by GF109203X or an antioxidant, Trolox. The present results support the idea that zinc influx activates PKC in the membrane, which contributes to free radical generation and neuronal death. As an increasing body of evidence suggests that zinc neurotoxicity is an important mechanism of pathological neuronal death, timely prevention of PKC activation after acute brain insult may prove useful in ameliorating this type of neuronal death.

Insulin-induced oxidative neuronal injury in cortical culture: mediation by induced N-methyl-D-aspartate receptors.

While effectively attenuating neuronal apoptosis in mouse cortical culture, insulin paradoxically induced neuronal necrosis with 48 h of exposure. The insulin neurotoxicity was blocked by an antioxidant but not by caspase inhibitors. Exposure to insulin led to tyrosine phosphorylation of the insulin receptor and the insulin-like growth factor-1 (IGF-1) receptor and activation of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3-kinase). Inhibitors of tyrosine kinase and PKC, but not PI3-kinase, attenuated the insulin neurotoxicity. Conversely, the inhibitor of PI3-kinase but not PKC reversed the antiapoptotic effect of insulin. Suggesting that the gene activity-dependent emergence of excitotoxicity contributed to insulin neurotoxicity, macromolecule synthesis inhibitors and N-methyl-D-aspartate (NMDA) antagonists blocked it. Consistently, exposure to insulin increased the level of the NR2A subunit of the NMDA receptor without much altering NR1 or NR2B levels. The present study suggests that insulin can be both neuroprotective and neurotoxic in the same cell system but by way of different signaling cascades.