Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR.

Ninety-three clinical isolates of vancomycin-resistant enterococci (VRE) collected from nine hospitals in Taiwan were examined for the presence of vanA, vanB, vanC1, or vanC2/vanC3 genes by a multiplex PCR. Forty-seven of these VRE isolates were vanA positive, 1 contained both vanC1 and vanA, 40 harboured vanB, 2 were vanC1, and 3 were identified to be vanC2/vanC3. Twenty-four vanA isolates were sensitive to teicoplanin and thus did not have a typical VanA phenotype. Five isolates with the VanC phenotype harboured vanB. None of the 40 clinically isolated vancomycin-susceptible E. faecium or E. faecalis and the vancomycin-resistant Leuconostoc and Pediococcus isolates were positive for any of the van genes. While performing nosocomial surveillance, VRE were isolated from 47 of 467 rectal swabs by culture. Compared with the conventional culture method, the sensitivity and specificity of the multiplex PCR for detecting and identifying vancomycin-resistance genes in enterococci directly from culture-positive broth were 97.9% and 100%, respectively. The results suggest that genotypic characterization of vancomycin-resistance is necessary for all clinical VRE isolates and that the multiplex PCR assay can be an alternative method for this purpose.

A comparison of methods for yeast identification including CHROMagar Candida, Vitek system YBC and a traditional biochemical method.

BACKGROUND: CHROMagar Candida (CAC) is a new chromogenic medium for the presumptive identification of clinically-important yeast isolates. A yeast biochemical card (YBC), a part of the Vitek system is an automatic method for the identification of clinically-important yeast isolates. We conducted a comparison of these two methods with a traditional biochemical method in order to choose a rapid and accurate technique for yeast identification. METHODS: All yeast isolates were inoculated onto Sabourand dextrose agar (SDA) and CAC, and incubated at 30 degrees C for 48 hours. All isolates were simultaneously tested using traditional biochemical methods and the yeast biochemical card from the Vitek system. RESULTS: We evaluated 235 yeast isolates from clinical specimens, including 89 Candida albicans, 47 Candida tropicalis, 43 Candida glabrata, six Trichosporon beigelii, and five Candida krusei in addition to 45 isolates of other yeast species. Isolates were presumptively identified on the basis of colony color and appearance on CAC medium. These observations were compared with a traditional biochemical yeast-identification method and also with YBC from the Vitek system. For five commonly-isolated species (Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei and Trichosporon beigelii), agreement among the CAC medium, YBC method and traditional biochemical method were 98.9% (187/189), 96.3% (182/189), 100% (189/189), respectively. CONCLUSIONS: From our comparison, the CAC medium is a convenient and economic method to identify five commonly-noted yeast species, and the YBC method warrants a greater cost and requires a longer period of time to obtain reliable results.

Phenytoin-induced asterixis--uncommon or under-diagnozed?

Asterixis is a recognized but uncommon clinical sign of phenytoin toxicity. A case is presented in which asterixis and acute cerebellar dysfunction occurred when the phenytoin levels reached the toxic range. It disappeared when the drug levels normalized. Asterixis is now classified as a form of negative myoclonus, which is characterized by irregular myoclonic lapses of posture. The neurochemistry and physiology of phenytoin causing asterixis has yet to be elucidated.

Characterization of a highly glycopeptide-resistant Enterococcus gallinarum isolate.

BACKGROUND AND PURPOSE: Adequate treatment of emergency infection involving antibiotic-resistant bacteria such as vancomycin-resistant Enterococcus requires a convergence of clinical and bacteriologic techniques. An isolate of Enterococcus gallinarum, designated as TSGH63, is known to be uncommonly vancomycin-resistant. This study investigated the genetic determinant for this unique characteristic. METHODS: After completing the conventional identification and sensitivity tests, the genomic content of E. gallinarum TSGH63 was extracted and analyzed by pulse-field electrophoresis. A set of specific primers for vanA, vanB, vanC1, and vanC2/C3 genes was then applied in a multiplex polymerase chain reaction (PCR) to differentiate its genetic content. To locate the determinant for high vancomycin resistance, the electrophoresis profile was further analyzed by Southern blot using the digoxigenin (DIG)-labeled vanA gene probe. Finally, interspecies transfer of the vancomycin-resistance determinant of E. gallinarum TSGH63 was tested by a conjugation experiment in vitro. RESULTS: A 50-kb plasmid was identified in the analysis of the genomic extract of E. gallinarum TSGH63 by pulse field electrophoresis. Using multiplex PCR, we demonstrated that E. gallinarum TSGH63 harbors a vanA gene in addition to a vanC1 gene. The DIG-labeled vanA gene-specific probe bound to the plasmid exclusively on the Southern blot. The plasmid-carried vanA gene, but not the vanC1 gene, was found to be transferable from TSGH63 to E. faecalis JH2-2 by conjugation in vitro. CONCLUSIONS: This is the first report of isolation of E. gallinarum with a high level of resistance to glycopeptides in Taiwan. The demonstrated interspecies transfer of the vancomycin-resistance gene highlights the importance of stringent control of the use of vancomycin.

Characterization of the first clinical isolate of vancomycin-resistant Enterococcus faecalis, AH803, in Taiwan.

We previously isolated a vancomycin-resistant strain of Enterococcus faecalis, designated AH803, from the sputum of a patient with pneumonia and bacteremia in Taiwan. AH803 was resistant to vancomycin (minimal inhibitory concentration, MIC = 512 micrograms/mL) but susceptible to teicoplanin (MIC = 8 micrograms/mL), and harbored the vanA gene but not the vanB gene. In this study, we further characterized E. faecalis AH803 and the plasmid it was found to contain. DNA from AH803 was analyzed for the presence of vanA and vanB resistance genes by polymerase chain reaction. The vancomycin resistant phenotype was transferable from AH803 to E. faecalis JH2-2, at a frequency of 4.8 x 10(-2). AH803 was also resistant to gentamicin and chloramphenicol, and these antibiotic resistance phenotypes cotransferred with vancomycin resistance. The genes responsible for resistance to all three antibiotics were located on a 42-kb conjugative plasmid (pBL101). This plasmid had the same restriction enzyme digestion patterns as Tn1546, found in pIP816 of E. faecalis BM4147. Epidemiologic studies of glycopeptide resistance should perhaps combine phenotypic and genotypic methods, rather than using phenotypic methods alone.

Clinical isolation of vancomycin-resistant Enterococcus faecalis in Taiwan.

We report the isolation of a vancomycin-resistant strain of Enterococcus faecalis, designated AH803, from a 76-year-old Taiwanese woman with pneumonia and bacteremia. This is the first documented clinical isolation of a vancomycin-resistant enterococcus in Taiwan. AH803 was repeatedly isolated from sputum specimens of the patient. AH803 had a high level of vancomycin (minimal inhibitory concentration, MIC = 512 micrograms/mL) and gentamicin (MIC > 2,000 micrograms/mL) resistance, but was susceptible to teicoplanin (MIC = 8 micrograms/mL) and ampicillin (MIC = 2 micrograms/mL). AH803 was shown by polymerase chain reaction to have the vanA gene, but not the vanB gene. Despite treatment efforts, the patient's condition continued to deteriorate. She requested to be discharged, against medical advice. The patient died at home the following day after discharge.

Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation.

Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca2+]i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca2+]i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca2+]i, which returned to near basal levels on restoration of O2-containing medium. Prolonged hypoxic conditions (> 60 min) caused irreversible elevation of [Ca2+]i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake ("point of no return"). Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca2+]i. The hypoxia-induced elevation of [Ca2+]i was also greatly attenuated if L-NAME (100 microM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca2+]i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the "point of no return," as characterized above, greatly potentiated [Ca2+]i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca2+]i and subsequent cell death, was not blocked by NOS inhibitor, L-NAME.

Changes in [Ca2+]i in cultured rat proximal tubular epithelium: an in vitro model for renal ischemia.

Two components of ischemia, oxygen deprivation and glycolytic inhibition, were studied in primary cultures of rat proximal tubular epithelial cells (PTE). Changes in cytosolic Ca2+ ([Ca2+]i) and its relationship to loss of mitochondrial membrane potential (delta psi m) and cell killing were characterized in single cells whereas ATP and LDH release were determined in populations of monolayer PTE. (1) Inhibition of mitochondrial respiration with KCN or anoxia resulted in little decrease in ATP or cell killing and slight change in [Ca2+]i over many hours. (2) Inhibition of respiration and glycolysis with anoxic HBSS minus glucose resulted in decreased ATP (54.4%) and cell killing (20%) during 5 h anoxic exposure. In all cases, but at highly variable times (113 +/- 62 min), [Ca2+]i initially rose to > 1 microM. In some cases it immediately dropped, stabilizing at about 500 nM for up to 1 h and rising again just prior to cell death. (3) Inhibition with anoxia + 1 mM IAA resulted in rapid depletion of ATP and cell killing, with increases in [Ca2+]i to > 1 microM by 20 +/- 2 min. (4) Depletion of glycolytic metabolites by depriving cells of substrate for 12 h (in HBSS minus glucose) before subjecting to anoxia minus glucose resulted in increases in [Ca2+]i at 40 +/- 17 min followed by cell killing. (5) Injury with anoxic HBSS minus glucose was reversed by reaeration before or during the initial rise in [Ca2+]i. Later reaeration resulted in rapid cell killing. In all cases, delta psi m was dissipated only after [Ca2+]i was significantly elevated.