Discovery of a potent phenolic N1-benzylidene- pyridinecarboxamidrazone selective against Gram-positive bacteria

As part of a study into antimycobacterial compounds a set of phenolic N1-benzylidene-pyridinecarboxamidrazones was prepared and evaluated. This report describes the unexpected discovery of a potent compound with a pronounced selectivity for Gram-positive bacteria over Gram-negative micro-organisms. In addition, this compound is active against various drug-resistant Gram-positive bacteria. © 2005 Elsevier Ltd. All rights reserved.

In vitro activities of novel oxapenems, alone and in combination with ceftazidime, against gram-positive and gram-negative organisms

Four novel oxapenem compounds (i.e., AM-112, AM-113, AM-114, and AM-115) were investigated for their β-lactamase inhibitory activity against a panel of isolated class A, C, and D enzymes, which included expanded-spectrum β-lactamase enzymes (ESBLs). The oxapenems were potent β-lactamase inhibitors. Activity varied within the group, with AM-113 and AM-114 proving to be the most active compounds. The 50% inhibitory concentrations for these agents were up to 100,000-fold lower than that of clavulanic acid against class C and D enzymes. As a group, the oxapenems were more potent than clavulanic acid against enzymes from all classes. The ability of these compounds to protect ceftazidime from hydrolysis by β-lactamase-producing strains was evaluated by MIC tests that combined ceftazidime and each oxapenem in a 1:1 or 2:1 ratio. The oxapenems markedly reduced the MICs for ceftazidime against class C hyperproducing strains and strains producing TEM- and SHV-derived ESBLs. There was little difference between the activity of 1:1 and 2:1 combinations of ceftazidime and oxapenem. The oxapenems failed to enhance the activity of ceftazidime against derepressed AmpC-producing Pseudomonas aeruginosa strains.

The postantibiotic effect of the carbapenem antibiotics on gram-negative bacteria

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.

A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

Toward bacterial protein sub-cellular location prediction:single-class discrimminant models for all gram- and gram+ compartments

Based on Bayesian Networks, methods were created that address protein sequence-based bacterial subcellular location prediction. Distinct predictive algorithms for the eight bacterial subcellular locations were created. Several variant methods were explored. These variations included differences in the number of residues considered within the query sequence - which ranged from the N-terminal 10 residues to the whole sequence - and residue representation - which took the form of amino acid composition, percentage amino acid composition, or normalised amino acid composition. The accuracies of the best performing networks were then compared to PSORTB. All individual location methods outperform PSORTB except for the Gram+ cytoplasmic protein predictor, for which accuracies were essentially equal, and for outer membrane protein prediction, where PSORTB outperforms the binary predictor. The method described here is an important new approach to method development for subcellular location prediction. It is also a new, potentially valuable tool for candidate subunit vaccine selection.

Constructive updating/downdating of oblique projectors:a generalization of the Gram-Schmidt process

A generalization of the Gram-Schmidt procedure is achieved by providing equations for updating and downdating oblique projectors. The work is motivated by the problem of adaptive signal representation outside the orthogonal basis setting. The proposed techniques are shown to be relevant to the problem of discriminating signals produced by different phenomena when the order of the signal model needs to be adjusted. © 2007 IOP Publishing Ltd.

Identification of DNA-binding proteins by incorporating evolutionary information into pseudo amino acid composition via the top-n-gram approach

DNA-binding proteins are crucial for various cellular processes and hence have become an important target for both basic research and drug development. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to establish an automated method for rapidly and accurately identifying DNA-binding proteins based on their sequence information alone. Owing to the fact that all biological species have developed beginning from a very limited number of ancestral species, it is important to take into account the evolutionary information in developing such a high-throughput tool. In view of this, a new predictor was proposed by incorporating the evolutionary information into the general form of pseudo amino acid composition via the top-n-gram approach. It was observed by comparing the new predictor with the existing methods via both jackknife test and independent data-set test that the new predictor outperformed its counterparts. It is anticipated that the new predictor may become a useful vehicle for identifying DNA-binding proteins. It has not escaped our notice that the novel approach to extract evolutionary information into the formulation of statistical samples can be used to identify many other protein attributes as well.

Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.