Mechanism of action of the novel aminomethylcycline antibiotic omadacycline.

Omadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistant Staphylococcus aureus (MRSA), ß-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae, Haemophilus influenzae, and Legionella. In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is active in vitro in the presence of the ribosomal protection protein Tet(O).

In vitro and in vivo antibacterial activities of omadacycline, a novel aminomethylcycline.

Omadacycline is the first intravenous and oral 9-aminomethylcycline in clinical development for use against multiple infectious diseases including acute bacterial skin and skin structure infections (ABSSSI), community-acquired bacterial pneumonia (CABP), and urinary tract infections (UTI). The comparative in vitro activity of omadacycline was determined against a broad panel of Gram-positive clinical isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Lancefield groups A and B beta-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae (PRSP), and Haemophilus influenzae (H. influenzae). The omadacycline MIC90s for MRSA, VRE, and beta-hemolytic streptococci were 1.0 µg/ml, 0.25 µg/ml, and 0.5 µg/ml, respectively, and the omadacycline MIC90s for PRSP and H. influenzae were 0.25 µg/ml and 2.0 µg/ml, respectively. Omadacycline was active against organisms demonstrating the two major mechanisms of resistance, ribosomal protection and active tetracycline efflux. In vivo efficacy of omadacycline was demonstrated using an intraperitoneal infection model in mice. A single intravenous dose of omadacycline exhibited efficacy against Streptococcus pneumoniae, Escherichia coli, and Staphylococcus aureus, including tet(M) and tet(K) efflux-containing strains and MRSA strains. The 50% effective doses (ED50s) for Streptococcus pneumoniae obtained ranged from 0.45 mg/kg to 3.39 mg/kg, the ED50s for Staphylococcus aureus obtained ranged from 0.30 mg/kg to 1.74 mg/kg, and the ED50 for Escherichia coli was 2.02 mg/kg. These results demonstrate potent in vivo efficacy including activity against strains containing common resistance determinants. Omadacycline demonstrated in vitro activity against a broad range of Gram-positive and select Gram-negative pathogens, including resistance determinant-containing strains, and this activity translated to potent efficacy in vivo.

Red wine prevents the postprandial increase in plasma cholesterol oxidation products: a pilot study.

Moderate wine consumption has been shown to lower cardiovascular risk. One of the mechanisms could involve the control of postprandial hyperlipaemia, a well-defined risk factor for atherosclerosis, reasonably by reducing the absorption of lipid oxidised species from the meal. The objective of the present study was to investigate whether wine consumption with the meal is able to reduce the postprandial increase in plasma lipid hydroperoxides and cholesterol oxidation products, in human subjects. In two different study sessions, twelve healthy volunteers consumed the same test meal rich in oxidised and oxidisable lipids (a double cheeseburger), with 300 ml of water (control) or with 300 ml of red wine (wine). The postprandial plasma concentration of cholesterol oxidation products was measured by GC-MS. The control meal induced a significant increase in the plasma concentration of lipid hydroperoxides and of two cholesterol oxidation products, 7-ß-hydroxycholesterol and 7-ketocholesterol. The postprandial increase in lipid hydroperoxides and cholesterol oxidation products was fully prevented by wine when consumed with the meal. In conclusion, the present study provides evidence that consumption of wine with the meal could prevent the postprandial increase in plasma cholesterol oxidation products.

Chlamydia pneumoniae induces T cell apoptosis through glutathione redox imbalance and secretion of TNF-alpha.

Chlamydia pneumoniae persistent infection has been implicated in the pathogenesis of several chronic inflammatory diseases including atherosclerosis, and we hypothesized that modulation of the apoptosis of macrophages and/or T cells by C. pneumoniae infection may contribute to the development of such diseases. We therefore evaluated apoptosis, cytokine response, and redox status in human primary T cells and macrophages infected with C. pneumoniae. In addition, co-cultures of T cells and macrophages infected with C. pneumoniae were also carried out. Apoptosis, and levels of glutathione (GSH), glutathione disulfide (GSSG), and tumour necrosis factor (TNF)-alpha were measured by flow cytometry, high performance liquid chromatography and enzyme-linked immunosorbent assay. C. pneumoniae induced apoptosis in T cells as well as in co-cultures of T cells and infected macrophages by marked decrease in GSH/GSSG ratio and increased production of TNF-alpha, respectively. The results demonstrate that interaction of C. pneumoniae with T cells and/or macrophages characterized by interference with redox status, and secretion of tumour necrosis factor-alpha culminates in the induction of T cell apoptosis and survival of infected macrophages. In conclusion, the inappropriate T cell response against C. pneumoniae and survival of infected macrophages could explain the persistence of this intracellular obligate pathogen in the host-organism; it may contribute to the development of chronic inflammatory diseases, although further studies are needed to clarify such a complex mechanism.

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress.

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.

Impact of simple screening criteria on utilization of low-yield bacterial stool cultures in a Children's Hospital.

OBJECTIVE: To determine diagnostic yield of stool cultures for Salmonella, Shigella, Campylobacter jejuni, Yersinia enterocolitica, and Escherichia coli O157:H7 (SSCYE) among hospitalized children and to develop guidelines for appropriate use of these tests. Setting. Tertiary care pediatric hospital. DESIGN: Computerized records from the Microbiology Laboratory from January 1992 to December 1996 were reviewed retrospectively to collect data on the number of stool cultures performed in inpatients and outpatients, the length of hospital stay at the time cultures were sent, and diagnostic yield of cultures in hospitalized patients. A detailed review of medical records of all patients with a stool pathogen isolated after 3 days of hospitalization was also undertaken. The results from this retrospective analysis were used to develop guidelines to reduce unwarranted stool cultures and to educate medical care providers in the appropriate use of these tests. The impact of these guidelines on reduction in the volume of stool cultures performed on hospitalized patients was measured prospectively from January 1998 to June 1998. RESULTS: A total of 27 110 stool cultures for SSCYE were performed in the 5-year study period. Of the 14 125 cultures from inpatients, 174 (1.2%) were positive. Among the cultures from inpatients, 9378 (66%) were from patients hospitalized for >3 days. Only 13 (.14%) were positive. Of these 13 cultures, 4 represented nosocomial infections, whereas the remaining 9 cultures either were sent to document clearance from a patient known previously to be infected with an enteric pathogen (7), or were attributed to delayed testing in individuals admitted with a diarrheal illness (2). Introduction of guidelines to reject all SSCYE cultures from patients hospitalized for >3 days who did not meet specified criteria was associated with an overall reduction of 689 (43%) in the volume of tests performed in the 6-month period evaluated. This included 497 fewer cultures ordered and 192 cultures that were ordered but rejected because screening criteria were not met. Only 11 (5.4%) of 203 cultures sent >3 days after admission were processed because they met clinical criteria for testing. None were positive. Estimated cost savings were $50 163/year. CONCLUSIONS: Stool cultures for SSCYE among hospitalized patients have very low diagnostic yield and are extremely overutilized. Simple guidelines, such as rejecting (with few exceptions) cultures from patients hospitalized for >3 days, can reduce substantially such unnecessary testing.

Impact of rapid antigen tests for group A streptococcal pharyngitis on physician use of antibiotics and throat cultures.

Using case scenarios and an interview guided by a decision tree diagram, the clinical strategies of 150 physicians (50 private pediatricians, 50 health maintenance organization pediatricians and 50 pediatric residents) were assessed for the management of pharyngitis under three conditions: (1) no rapid antigen detection test available for diagnosing Group A streptococcal disease; (2) antigen test result available in 20 minutes; and (3) antigen test result available in 4 hours. The sensitivity of the antigen test was designated as 0.95 if the growth of rare or few Group A streptococci on throat culture was discounted and 0.82 if any growth was considered significant, and the specificity was set at 0.98. In a standardized pediatric case with a prior probability of Group A streptococcal disease of 0.58, 84% of clinicians would order a 20-minute test and 39% would order a 4-hour test. The 20-minute test would reduce throat culture use by 54%, reduce the proportion of patients exposed to antibiotics from 86% to 65% and reduce total antibiotic treatment days by 13.8%. Effects would be less pronounced for a low probability case or if results of antigen testing were not available for 4 hours. Provided a test with a documented high sensitivity and specificity were used, a rapid antigen test with results promptly available would substantially reduce throat culture use and unnecessary antibiotic exposures in children with a moderate prior probability of streptococcal disease.

Performance of a solid phase enzyme immunoassay for detection of group A streptococci in a pediatric office laboratory as refereed by a hospital laboratory.

We evaluated the performance of a new rapid solid phase enzyme immunoassay, SUDS Group A Strep (MUREX Corp., Norcross, GA) for the detection of Group A beta-hemolytic streptococci in a pediatric office practice. Duplicate throat swabs were obtained from 341 children with pharyngitis. One swab was used in the SUDS test and the other was cultured in the office laboratory. Office SUDS and culture (sheep blood agar plate, aerobic 24-hour incubation) were compared with culture using reference techniques (sheep blood agar plate, anaerobic 48-hour incubation) in a hospital laboratory. Compared with hospital laboratory culture, the sensitivity of office SUDS (73.8%) was superior to that of office culture (66.6%) at P = 0.05. Specificities were 93.1 and 98.6%, respectively; positive predictive values were 86.1 and 96.6%; and negative predictive values were 85.9 and 83.5%. The sensitivity and specificity of SUDS compared with office culture were 88.5 and 87.8%, respectively, but would have been 93 and 94% had hemolyzed media not been used on several occasions in the office culture procedure. We conclude that SUDS Group A Strep was significantly more sensitive than throat cultures as performed in a typical pediatric practice although the performance of office cultures could have been improved by standard quality control techniques.

Detection of a decarboxylated dimer of aminoethylcysteine ketimine in bovine cerebellum.

Aminoethylcysteine ketimine is a sulfur-containing cyclic compound produced by the enzymatic alpha-deamination of the parent aminoethylcysteine that has been detected in bovine brain and cerebellum. Aminoethylcysteine ketimine is known to dimerize spontaneously and easily lose one carboxyl group. This decarboxylated compound, simply named the dimer, has been recently detected in normal human urine. In this article we provide evidence on the occurrence of the dimer in the bovine cerebellum.

Facilitated detection of antibiotic-resistant Pseudomonas in cystic fibrosis sputum using homogenized specimens and antibiotic-containing media.

Sputa from 30 patients with cystic fibrosis (CF) were cultured on routine and selective media plus three Mueller-Hinton antibiotic resistance screening plates containing tobramycin (5 micrograms/ml), azlocillin (100 micrograms/ml), and ticarcillin (100 micrograms/ml). In addition to direct semiquantitative plating, samples were homogenized for semiquantitative and quantitative culture. Blood agar plates from direct semiquantitative and homogenized semiquantitative cultures were then replica plated onto the antibiotic screening plates. Homogenized semiquantitative and quantitative cultures both detected more Pseudomonas aeruginosa strains than direct semiquantitative plating (103 versus 85 strains), including more antibiotic-resistant strains. Antibiotic screening media facilitated isolation of resistant strains and decreased detection time by 24 hr. Of the 103 strains on homogenized semiquantitative and quantitative cultures isolated before replica plating, 13 (13%) were tobramycin-resistant, 67 (65%) were ticarcillin-resistant, and 42 (41%) were azlocillin-resistant; 13 of 30 cultures (43%) had at least one tobramycin-resistant organism before replica plating. Replica plating detected an additional seven tobramycin-resistant and nine ticarcillin- or azlocillin-resistant strains in seven patients. Homogenization, antibiotic screening media, and replica plating enhance recognition of antibiotic-resistant strains in CF sputum.

Antioxidant properties of the decarboxylated dimer of aminoethylcysteine ketimine: assessment of its ability to scavenge peroxynitrite.

The natural sulfur compound aminoethylcysteine ketimine decarboxylated dimer (AECK dimer) has been investigated for its ability to act as peroxynitrite scavenger. It has been found that the product efficiently protects against the nitration of tyrosine and the inactivation of alpha1-antiproteinase by peroxynitrite. The tyrosine nitration can be completely prevented by 100 microM AECK dimer which appears as effective as the antioxidants glutathione and N-acetylcysteine. The AECK dimer was also found to limit surface charge alteration of low density lipoprotein induced by peroxynitrite. These findings indicate that the AECK dimer is a strong protective agent against peroxynitrite damage and that it could play an important role in the defence against oxidative stress in human diseases.

Determination of aminoethylcysteine ketimine decarboxylated dimer in human plasma and cultured cells by high-performance liquid chromatography with electrochemical detection.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural compound with antioxidant properties of a new family of sulfur-containing amino acids. It has been detected in human urine and plasma, in mammalian cerebellum and, more recently, in dietary vegetables. In the present study, a simple, highly sensitive method using a high-performance liquid chromatography system with electrochemical detection (ECD) has been developed. The method showed excellent precision and accuracy. It has been found to be about 100-fold more sensitive than gas chromatographic method and 2000-fold more sensitive in respect to the liquid chromatography method with UV detection. The method showed the required features of specificity and sensitivity to detect aminoethylcysteine ketimine decarboxylated dimer in human plasma and in cultured cells after in vitro supplementation.

Effects of traditional and ultrasonic liposuction on adipose tissue: a biochemical approach.

Little is known about the interaction of ultrasonic liposculpture with fat tissue. The surgical technique is well established and its clinical effects are satisfactory. However, the in vivo effects on adipose tissue remain to be determined. Previous studies have shown that ultrasound waves break fat cells. The purpose of this study was to ascertain whether ultrasound waves can cause the release of fatty acids from the molecular structure of triglycerides. A double-blind study was designed with samples obtained from traditional and ultrasonic liposuction of an equivalent area in the same patient. Samples were checked for triglycerides and for free fatty acids. Triglyceride values were always higher in the sample that had undergone ultrasonic procedure. No significant differences were observed between the free fatty acid chromatograms of the two kinds of samples analyzed. Data showed that no changes occurred in the triglyceride molecule when using ultrasound waves in the experimental conditions.

Aminoethylcysteine ketimine decarboxylated dimer detected in normal human urine by gas-liquid chromatography, selected-ion monitoring and mass spectrometry.

Aminoethylcysteine ketimine is a biochemical product known to be converted spontaneously in the decarboxylated dimer. Since the ketimine has been detected in a mammalian brain, it was assumed that also the dimer could be present in the mammalian body and eventually excreted in the urine. Using human urine as the biological source, an extract was prepared which, submitted to gas-liquid chromatography, selected-ion monitoring and mass spectrometry, indicated the presence of the dimer.

Osteomyelitis caused by Capnocytophaga ochracea.

A 13-year-old boy is reported with osteomyelitis of the greater trochanter caused by a facultative anaerobic organism (Capnocytophaga ochracea) not known to cause infections in normal immunocompetent individuals, nor implicated in bone or joint infections even in compromised hosts. There remains a need to consider unusual organisms and to obtain accurate bacteriologic data in all cases of osteomyelitis.

Bacterial colonization of neonates admitted to an intensive care environment.

In order to elucidate some of the factors responsible for the high rate of nosocomial infection associated with neonatal intensive care, we studied bacterial colonization in 63 infants admitted to a neonatal intensive care unit. In a six-month period, cultures of nose, throat, umbilicus, and stool were obtained on admission and every three days from all infants staying in the NICU greater than or equal to 3 days. Study infants did not develop "normal" aerobic flora. Forty-eight percent of infants grew Escherichia coli from stool, but 52% had stool colonization with Klebsiella, Enterobacter, or Citrobacter, the only other Enterobacteriaceae encountered. KEC were also isolated from throat, nose, and umbilicus in 22%, 22%, and 24% of patients, respectively. The risk of stool colonization with KEC increased with duration of hospitalization: 2% of infants were colonized on admission, 60% after 15 days, and 91% after 30 days. Stool colonization with E. coli seemed to protect infants from colonization with other gram-negative bacilli. Thirteen of 20 infants, however, developed pharyngeal GNB colonization in spite of pre-existing abundant growth of alpha streptococci. Antibiotic therapy for greater than 3 days was associated with the isolation of KEC in stool and GNB in the throat, but birth weight less than 2,500 gm and lack of breast milk feedings were not.

Changes in intestinal flora of farm personnel after introduction of a tetracycline-supplemented feed on a farm.

A prospective study was undertaken to determine whether feeding farm animals antibiotics in feed caused changes in the intestinal bacterial flora of farm dwellers and their neighbors. Chickens were fed tetracycline-supplemented feed (tet-feed), and, as expected, within one week their intestinal flora contained almost entirely tetracycline-resistant organisms. Increased numbers of resistant intestinal bacteria also appeared, but more slowly, in farm members, but not their neighbors. Within five and six months, 31.3 per cent of weekly fecal samples from farm dwellers contained greater than 80 per cent tetracycline-resistant bacteria as compared to 6.8 per cent of the samples from the neighbors (P less than 0.001). Seven of the 11 farm members, but only three of the 24 neighbors, had two or more fecal samples containing greater than 80 per cent tetracycline-resistant coliforms (P less than 0.01). These resistant bacteria contained transferable plasmids conferring multiple antibiotic resistances. Selective pressure by tet-feed for antibiotic-resistant bacteria in chickens extends to human beings in contact with chickens and the feed.

Enzymatically labelled nucleic acid (NA) probe assays for detection of Campylobacter spp. in human faecal specimens and in culture.

Seventy-two stock clinical isolates of C. jejuni were tested with the SNAP Culture Identification Test Kit (Molecular Biosystems, Inc., San Diego, CA), an enzyme labelled nucleic acid probe colony blot assay, and with the Campyslide (BBL Microbiology Systems, Cockeysville, MD), a latex agglutination test. The sensitivity and overall agreement of both the SNAP and Campyslide tests were 100% in comparison with standard culture and identification tests. In addition, 14 C. jejuni, two C. laridis, and one 'C. upsaliensis', and one C. hyointestinalis, all fresh isolates from acutely ill patients, were detected with both tests. Ninety-eight faecal specimens from acutely ill patients were tested within 48 h using the probe based SNAP Diagnostic Test Kit (Molecular Biosystems, Inc.). Results were compared with standard culture. The probe test detected 4/6 specimens positive by culture; sensitivity was 66%. However, the two culture positive specimens, undetected by the probe test, had pathogens present at levels well below the probe test sensitivity (10(5) CFU g-1 stool). Specificity of the probe test was 98% (90/92 negative specimens) compared to culture. The results of this study demonstrate the accuracy of enzyme labelled oligonucleotide probes in the identification of C. jejuni isolated on culture and suggest a potential role for such probes in the direct detection of campylobacter in clinical faecal specimens.

Catheter infection caused by Methylobacterium in immunocompromised hosts: report of three cases and review of the literature.

Three cases of catheter infection due to Methylobacterium extorquens are reported. Each patient had a history of acute leukemia and was immunocompromised; two had undergone bone marrow transplantation, and the third was receiving consolidation chemotherapy. All three patients survived after removal of the central venous catheter and antibiotic treatment. The clinical features of these cases are compared with those of the 12 previously reported cases of infection due to Methylobacterium species.