Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were > or =4 microg/ml or for which the teicoplanin MICs were > or =8 microg/ml and 24 control strains for which the vancomycin MICs were < or =2 microg/ml or for which the teicoplanin MICs were < or =4 microg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 microg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 microg/ml were noted for these isolates. Vitek results were 4 microg/ml for all strains for which the vancomycin MICs were > or =4 microg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either < or =2 or > or =16 microg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 microg of vancomycin per ml consistently differentiated those strains inhibited by 8 microg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.     

Metronidazole. A therapeutic review and update

The nitroimidazole antibiotic metronidazole has a limited spectrum of activity that encompasses various protozoans and most Gram-negative and Gram-positive anaerobic bacteria. Metronidazole has activity against protozoans like Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis, for which the drug was first approved as an effective treatment. Anaerobic bacteria which are typically sensitive are primarily Gram-negative anaerobes belonging to the Bacteroides and Fusobacterium spp. Gram-positive anaerobes such as peptostreptococci and Clostridia spp. are likely to test sensitive to metronidazole, but resistant isolates are probably encountered with greater frequency than with the Gram-negative anaerobes. Gardnerella vaginalis is a pleomorphic Gram-variable bacterial bacillus that is also susceptible to metronidazole. Helicobacter pylori has been strongly associated with gastritis and duodenal ulcers. Classic regimens for eradicating this pathogen have included metronidazole, usually with acid suppression medication plus bismuth and amoxicillin. The activity of metronidazole against anaerobic bowel flora has been used for prophylaxis and treatment of patients with Crohn's disease who might develop an infectious complication. Treatment of Clostridium difficile-induced pseudomembraneous colitis has usually been with oral metronidazole or vancomycin, but the lower cost and similar efficacy of metronidazole, coupled with the increased concern about imprudent use of vancomycin leading to increased resistance in enterococci, have made metronidazole the preferred agent here. Metronidazole has played an important role in anaerobic-related infections. Advantages to using metronidazole are the percentage of sensitive Gram-negative anaerobes, its availability as oral and intravenous dosage forms, its rapid bacterial killing, its good tissue penetration, its considerably lower chance of inducing C. difficile colitis, and expense. Metronidazole has notable effectiveness in treating anaerobic brain abscesses. Metronidazole is a cost-effective agent due to its low acquisition cost, its pharmacokinetics and pharmacodynamics, an acceptable adverse effect profile, and its undiminished antimicrobial activity. While its role as part of a therapeutic regimen for treating mixed aerobic/anaerobic infections has been reduced by newer, more expensive combination therapies, these new combinations have not been shown to have any therapeutic advantage over metronidazole. Although the use of metronidazole on a global scale has been curtailed by newer agents for various infections, metronidazole still has a role for these and other therapeutic uses. Many clinicians still consider metronidazole to be the 'gold standard' antibiotic against which all other antibiotics with anaerobic activity should be compared.     

All-trans-retinoic acid in acute promyelocytic leukemia

BACKGROUND: All-trans-retinoic acid induces complete remission in acute promyelocytic leukemia. However, it is not clear whether induction therapy with all-trans-retinoic acid is superior to chemotherapy alone or whether maintenance treatment with all-trans-retinoic acid improves outcome. METHODS: Three hundred forty-six patients with previously untreated acute promyelocytic leukemia were randomly assigned to receive all-trans-retinoic acid or daunorubicin plus cytarabine as induction treatment. Patients who had a complete remission received consolidation therapy consisting of one cycle of treatment identical to the induction chemotherapy, then high-dose cytarabine plus daunorubicin. Patients still in complete remission after two cycles of consolidation therapy were then randomly assigned to maintenance treatment with all-trans-retinoic acid or to observation. RESULTS: Of the 174 patients treated with chemotherapy, 120 (69 percent) had a complete remission, as did 124 of the 172 (72 percent) given all-trans-retinoic acid (P=0.56). When both induction and maintenance treatments were taken into account, the estimated rates of disease-free survival at one, two, and three years were 77, 61, and 55 percent, respectively, for patients assigned to chemotherapy then all-trans-retinoic acid; 86, 75, and 75 percent for all-trans-retinoic acid then all-trans-retinoic acid; 75, 60, and 60 percent for all-trans-retinoic acid then observation; and 29, 18, and 18 percent for chemotherapy then observation. By intention-to-treat analysis, the rates of overall survival at one, two, and three years after entry into the study were 75, 57, and 50 percent, respectively, among patients assigned to chemotherapy, and 82, 72, and 67 percent among those assigned to all-trans-retinoic acid (P= 0.003). CONCLUSIONS: All-trans-retinoic acid as induction or maintenance treatment improves disease-free and overall survival as compared with chemotherapy alone and should be included in the treatment of acute promyelocytic leukemia.     

Cytokine-mediated growth hormone release from cultured ovine pituitary cells

Previous studies have demonstrated that intravenous lipopolysaccharide (LPS) will increase concentrations of growth hormone (GH). One possible explanation for this may reside in the response of the pituitary to specific cytokines. This study sought to determine the effects of recombinant bovine tumor necrosis factor alpha (TNF), recombinant ovine (ro) interleukin-1alpha (IL-1alpha), roIL-1beta, ro interleukin-2 (IL-2), and ro gamma-interferon (INT) on GH release from cultured sheep pituitary cells. Sheep were sacrificed and pituitary cells cultured in DMEM with 10% fetal bovine serum for 3 days. On day 4, cells were washed and serum-free DMEM added to cells. IL-1alpha and IL-1beta were used at 0.2, 2 and 20 ng/ml and the remaining cytokines at 2, 20 and 200 ng/ml. Neither IL-2 nor INT had effects on basal or on GH-releasing hormone (GRH)-stimulated GH release. TNF inhibited GRH-stimulated GH release (p < 0.05). Both IL-1alpha and IL-1beta stimulated GH release from cultured pituitary cells at all doses tested (p < 0.01). Neither IL-1alpha nor IL-1beta had an effect on GRH-stimulated GH release. IL-1 effects were inhibited by H-89 (p < 0.05; a protein kinase A inhibitor) and by nifedipine (p < 0.05; a calcium channel blocker). Both of these mechanisms are central signal transduction mechanisms mediating GRH-stimulated GH release. IL-1-stimulated GH release is partially inhibited (p < 0.05) by lipoxygenase pathway blockers. Phorbol myristate acetate downregulation of protein kinase C did not alter IL-1-stimulated GH release. IL-1beta increased the content of both GH and GH mRNA in cultured sheep pituitary cells. We conclude that IL-1 produces a strong stimulus to GH release, which is mediated by calcium entry and protein kinase A activation. IL-1 also activates lipoxygenase pathways. This latter pathway as well as calcium entry were shown to mediate LPS stimulation of GH release from cultured pituitary cells. The similarity between IL-1 and LPS signal transduction suggests that LPS may activate pituitary production of IL-1 to produce the stimulus to GH. The lack of inhibitory effects of INT, TNF and IL-2 as opposed to what is seen in the rat may suggest a partial mechanism to explain the different effects of LPS on GH release between sheep and that seen in cattle and rats.     

Resistance to antibiotic and heavy metals of motile aeromonads from Chilean freshwater

In this work the resistance of 172 motile Aeromonas isolates recovered from raw drinking water supplies (56), irrigation waters (60) and runoff waters receiving sewage (56), to some antibiotics and heavy metals was investigated by agar diffusion and agar dilution methods. A high proportion of isolates from all water sources showed resistance to carbenicillin, erythromycin, streptomycin, cephradine and cadmium, and susceptibility to chloramphenicol, kanamycin, gentamicin, tetracycline, nalidixic acid, trimethoprim-sulphametoxazole and chromium. No amikacin-resistant Aeromonas were recovered. No relationship was found between antimicrobial resistance and Aeromonas species, with the exception of cephradine, that exhibited a significantly higher activity against the A. sobria isolates than the other Aeromonas species (P < 0.05). Moderately polluted waters showed lower antibiotic multiresistance and metal susceptibility than unpolluted and highly polluted ones. Although significant differences (P < 0.05), between resistance frequencies to erythromycin, carbenicillin, streptomycin and cephradine were found among isolates from different sources, the antimicrobial resistance patterns of aeromonads could not be related to the level of faecal pollution. These results indicate that aeromonads resistant to antibiotics and heavy metals are easily recovered from water sources in Chile, posing a potential public health risk.     

Bidirectional transfer between electrical and flurothyl kindling in mice: evidence for common processes in epileptogenesis

PURPOSE: This study sought to determine whether there was a transfer of seizure susceptibility between two models of epileptogenesis, electrical kindling and a newly described model of flurothyl kindling. In this study, we determined the effects of preexposure to one kindling agent on the seizure responsiveness to the other. METHODS: Mice were divided into three groups: (a) six mice (FLK) were kindled with flurothyl, rechallenged with flurothyl after a 28-day incubation phase, implanted with olfactory bulb (OB) electrodes, and electrically kindled; (b) six mice (ELK) were implanted with OB electrodes, electrically kindled to six stage 5 seizures, and given one flurothyl trial 3 days later and a second flurothyl trial after a 28-day incubation period; and (c) six mice (IMP) were implanted with OB electrodes, tested with flurothyl at the same times as the ELK group, and later electrically kindled. RESULTS: Mice that were previously kindled with flurothyl (FLK) had significantly faster electrical kindling rates to one stage 5 seizure or to six stage 5 seizures compared with animals in the ELK and IMP groups. Mice that were previously exposed to either electrical kindling or flurothyl kindling had significantly diminished latencies to generalized seizure onset (flurothyl-induced seizure thresholds) either before or after a 28-day incubation period compared with the IMP control mice. In addition, both the FLK and ELK groups had significantly increased percentages of mice expressing forebrain-brainstem seizures, compared with the IMP group, following either rechallenge with flurothyl after a 28-day incubation or focal electrical kindling. CONCLUSIONS: These findings indicate a near-complete bidirectional transfer between these electrical and flurothyl kindling models. Mice that were previously exposed to either electrical or flurothyl kindling have increased seizure susceptibilities and altered seizure phenotypes when exposed to the other seizure paradigm. Overall, these studies indicate that previous seizures are the critical determinant of the bidirectional transfer of seizure susceptibility observed, and not the electrical or pharmacologic properties of the original kindling agent. Finally, the observation of near identity in transfer characteristics between electrical and flurothyl kindling models suggests that the proepileptogenic processes initiated by exposure to either model are similar.     

Centripetal cholesterol flux to the liver is dictated by events in the peripheral organs and not by the plasma high density lipoprotein or apolipoprotein A-I concentration

The major net flux of cholesterol in the intact animal or human is from the peripheral organs to the liver. This flux is made up of cholesterol that is either synthesized in these peripheral tissues or taken up as lipoprotein cholesterol. This study investigates whether it is the concentration of apolipoprotein (apo) A-I or high density lipoprotein in the plasma that determines the magnitude of this flux or, alternatively, whether events within the peripheral cells themselves regulate this important process. In mice that lack apoA-I and have very low concentrations of circulating high density lipoprotein, it was found that there was no accumulation of cholesterol in any peripheral organ so that the mean sterol concentration in these tissues was the same (2208 +/- 29 mg/kg body weight) as in control mice (2176 +/- 50 mg/kg). Furthermore, by measuring the rates of net cholesterol acquisition in the peripheral organs from de novo synthesis and uptake of low density lipoprotein, it was demonstrated that the magnitude of centripetal sterol movement from the peripheral organs to the liver was virtually identical in control animals (78 +/- 5 mg/day per kg) and in those lacking apoA-I (72 +/- 4 mg/day per kg). These studies indicate that the magnitude of net sterol flux through the body is not related to the concentration of high density lipoprotein or apolipoprotein A-I in the plasma, but is probably determined by intracellular processes in the peripheral organs that dictate the rate of movement of cholesterol from the endoplasmic reticulum to the plasma membrane.     

Obstructive sleep apnea during pregnancy resulting in pulmonary hypertension

Obesity is known to increase maternal morbidity and mortality. We describe a case of obstructive sleep apnea due to obesity and discuss our treatment of the resulting pulmonary hypertension. A patient was transferred to our hospital at 29 weeks' gestation with severe anasarca and more than a 100-pound weight gain during pregnancy. Pulmonary hypertension due to obstructive sleep apnea was diagnosed. The patient was treated with nasal continuous positive airway pressure (CPAP) during sleep and remained in the hospital the remainder of her pregnancy. She had a massive spontaneous diuresis during her hospital stay and lost more than 100 pounds. She was delivered at term via cesarean section because of transverse lie. Preoperative hemodynamic monitoring confirmed the diagnosis of pulmonary hypertension. This represents the first case in the literature of obstructive sleep apnea leading to pulmonary hypertension in pregnancy. This patient responded well to nasal CPAP as evident by the massive diuresis and good maternal outcome.     

Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS

The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.