Evaluation of the microbiota-sparing properties of the anti-staphylococcal antibiotic afabicin.

BACKGROUND: Antibiotic use is associated with collateral damage to the healthy microbiota. Afabicin is a first-in-class prodrug inhibitor of the FabI enzyme that, when converted to the pharmacologically active agent afabicin desphosphono, demonstrates a staphylococcal-specific spectrum of activity. An expected benefit of highly targeted antibiotics such as afabicin is microbiome preservation. OBJECTIVES: To compare the effects of oral treatment with afabicin and standard-of-care antibiotics upon the murine gut microbiota, and to assess the effects of oral afabicin treatment on the human gut microbiota. METHODS: Gut microbiota effects of a 10 day oral course of afabicin treatment were monitored in mice and compared with clindamycin, linezolid and moxifloxacin at human-equivalent dose levels using 16S rDNA sequencing. Further, the gut microbiota of healthy volunteers was longitudinally assessed across 20 days of oral treatment with afabicin 240 mg twice daily. RESULTS: Afabicin treatment did not significantly alter gut microbiota diversity (Shannon H index) or richness (rarefied Chao1) in mice. Only limited changes to taxonomic abundances were observed in afabicin-treated animals. In contrast, clindamycin, linezolid and moxifloxacin each caused extensive dysbiosis in the murine model. In humans, afabicin treatment was not associated with alterations in Shannon H or rarefied Chao1 indices, nor relative taxonomic abundances, supporting the findings from the animal model. CONCLUSIONS: Oral treatment with afabicin is associated with preservation of the gut microbiota in mice and healthy subjects.

Carbapenemase-producing Enterobacteriaceae in Australian hospitals: outcome of point-prevalence screening in high-risk wards.

Carbapenemase-producing Enterobacteriaceae (CPE) infections are increasingly reported in Australian hospitals, but prevalence is unknown. In 2016, Victorian hospitals conducted CPE point-prevalence surveys in high-risk wards (intensive care, haematology, transplant). Forty-three hospitals performed 134 surveys, with 1839/2342 (79%) high-risk patients screened. Twenty-four surveys were also performed in other wards. Inability to obtain patient consent was the leading reason for non-participation. In high-risk wards, no CPE cases were detected; three cases were identified in other wards. Since there is low prevalence in high-risk wards, continuous screening is not recommended. Targeted screening may be enhanced by review of patient consent processes.

Vancomycin-intermediate Staphylococcus aureus isolates are attenuated for virulence when compared with susceptible progenitors.

OBJECTIVES: Vancomycin-intermediate Staphylococcus aureus (VISA) is associated with genetic changes that may also impact upon pathogenicity. In the current study, we compared the virulence of clinical VISA strains with their isogenic vancomycin-susceptible progenitors (VSSA). METHODS: Production of the critical virulence protein, α toxin, was assessed using Western blot analysis and was correlated to agr activity using a bioluminescent agr-reporter. Cytotoxicity and intracellular persistence were compared ex vivo for VSSA and VISA within non-professional phagocytes (NPP). Virulence and host immune responses were further explored in vivo using a murine model of bacteraemia. RESULTS: VISA isolates produced up to 20-fold less α toxin compared with VSSA, and this was corroborated by either loss of agr activity due to agr mutation, or altered agr activity in the absence of mutation. VISA were less cytotoxic towards NPP and were associated with enhanced intracellular persistence, suggesting that NPP may act as a reservoir for VISA. Infection with VSSA strains produced higher mortality in a murine bacteraemia model (≥90% 7-day mortality) compared with infection with VISA isolates (20% to 50%, p <0.001). Mice infected with VISA produced a dampened immune response (4.6-fold reduction in interleukin-6, p <0.001) and persistent organ bacterial growth was observed for VISA strains out to 7 days. CONCLUSIONS: These findings highlight the remarkable adaptability of S. aureus, whereby, in addition to having reduced antibiotic susceptibility, VISA alter the expression of pathogenic factors to circumvent the host immune response to favour persistent infection over acute virulence.

Significant reduction in vancomycin-resistant enterococcus colonization and bacteraemia after introduction of a bleach-based cleaning-disinfection programme.

BACKGROUND: Vancomycin-resistant enterococcus (VRE) colonization and infection have increased at our hospital, despite adherence to standard VRE control guidelines. AIM: We implemented a multi-modal, hospital-wide improvement programme including a bleach-based cleaning-disinfection programme ('Bleach-Clean'). VRE colonization, infection and environmental contamination were compared pre and post implementation. METHODS: The programme included a new product (sodium hypochlorite 1000 ppm + detergent), standardized cleaning-disinfection practices, employment of cleaning supervisors, and modified protocols to rely on alcohol-based hand hygiene and sleeveless aprons instead of long-sleeved gowns and gloves. VRE was isolated using chromogenic agar and/or routine laboratory methods. Outcomes were assessed during the 6 months pre and 12 months post implementation, including proportions (per 100 patients screened) of VRE colonization in high-risk wards (HRWs: intensive care, liver transplant, renal, haematology/oncology); proportions of environmental contamination; and episodes of VRE bacteraemia throughout the entire hospital. FINDINGS: Significant reductions in newly recognized VRE colonizations (208/1948 patients screened vs 324/4035, a 24.8% reduction, P = 0.001) and environmental contamination (66.4% reduction, P = 0.012) were observed, but the proportion of patients colonized on admission was stable. The total burden of inpatients with VRE in the HRWs also declined (median percentage of colonized inpatients per week, 19.4% vs 17.3%, P = 0.016). Hospital-wide VRE bacteraemia declined from 14/2935 patients investigated to 5/6194 (83.1% reduction; P < 0.001), but there was no change in vancomycin-susceptible enterococcal bacteraemia (P = 0.54). CONCLUSION: The Bleach-Clean programme was associated with marked reductions in new VRE colonizations in high-risk patients, and VRE bacteraemia across the entire hospital. These findings have important implications for VRE control in endemic healthcare settings.

Solid-phase synthesis of peptidomimetic inhibitors for the hepatitis C virus NS3 protease.

The NS3 serine protease enzyme of the hepatitis C virus (HCV) is essential for viral replication. Short peptides mimicking the N-terminal substrate cleavage products of the NS3 protease are known to act as weak inhibitors of the enzyme and have been used as templates for the design of peptidomimetic inhibitors. Automated solid-phase synthesis of a small library of compounds based on such a peptidomimetic scaffold has led to the identification of potent and highly selective inhibitors of the NS3 protease enzyme.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

NMR line-broadening and transferred NOESY as a medicinal chemistry tool for studying inhibitors of the hepatitis C virus NS3 protease domain.

This work describes the use of NMR as a medicinal chemistry tool for better understanding the binding characteristics of inhibitors of the HCV NS3 protease. The protease-bound structure of a tetrapeptide-like inhibitor that has an acid C-terminus, a norvaline at P1 and a naphthylmethoxy proline at P2 is described. Conformational comparisons are made with a similar compound having a 1-amino-cyclopropylcarboxylic acid at P1 and with a hexapeptide inhibitor. Differences between the free and bound states are identified. 19F NMR also helped in determining that a single complex is observed when an inhibitor is added to the protease at a 1:1 ratio.

2',6'-Dimethylphenoxyacetyl: a new achiral high affinity P(3)-P(2) ligand for peptidomimetic-based HIV protease inhibitors.

Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1-2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors.

Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain.

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Phosphotyrosine-containing dipeptides as high-affinity ligands for the p56lck SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.

Burn wound excision and massive blood transfusion did not affect perioperative vancomycin levels.

INTRODUCTION: The effect of burns surgery that requires intraoperative transfusion of five or more units of blood on serum vancomycin levels was assessed. METHODS: Serum vancomycin levels were measured 10 min and 6 h after vancomycin administration with surgery being performed in the interval. The following day the same dose of vancomycin was given and serum vancomycin levels measured at the same times without intervening surgery. RESULTS: Thirteen operations involving nine patients who required a mean blood transfusion of 9.2 units were studied. There was very little difference between 10-min levels, 6-h levels and the change over interval (absolute and percentage) on the day of surgery and the following day. The recorded serum levels were often at the lower end of the desired range, especially in patients who underwent the larger operations. This was the case on both day of surgery and the control day. CONCLUSIONS: Large volume blood loss and replacement during burns surgery did not significantly affect perioperative vancomycin levels. 1998 Elsevier Science Ltd for ISBI.

Human cytomegalovirus protease complexes its substrate recognition sequences in an extended peptide conformation.

Substrate hydrolysis by human cytomegalovirus (HCMV) protease is essential to viral capsid assembly. The interaction of HCMV protease and the N-terminal cleavage products of the hydrolysis of R- and M-site oligopeptide substrate mimics (R and M, respectively, which span the P9-P1 positions) was studied by NMR methods. Protease-induced differential line broadening indicated that ligand binding is mediated by the P4-P1 amino acid residues of the peptides. A well-defined extended conformation of R from P1 through P4 when complexed to HCMV protease was evidenced by numerous transferred nuclear Overhauser effect (NOE) correlations for the peptide upon addition of the enzyme. NOE cross-peaks between the P4 and P5 side chains placing these two groups in proximity indicated a deviation from the extended conformation starting at P5. Similar studies carried out for the M peptide also indicated an extended peptide structure very similar to that of R, although the conformation of the P5 glycine could not be established. No obvious variation in structure between bound R and M (notably at P4, where the tyrosine of the R-site has been suggested to play a key role in ligand binding) could be discerned that might explain the observed differences in processing rates between R- and M-sequences. Kinetic studies, utilizing R- and M-site peptide substrates for which the P5 and P4 residues were separately exchanged, revealed that these positions had essentially no influence on the specificity constants (kcat/KM). In sharp contrast, substitution of the P2 residue of an M-site peptide changed its specificity constant to that of an R-site peptide substrate, and vice versa.

beta-Lactam derivatives as inhibitors of human cytomegalovirus protease.

The development of novel monobactam inhibitors of HCMV protease incorporating a carbon side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at the C-3 position of the beta-lactam ring gave an increase in enzymatic activity and in stability; however, a lack of selectivity against other serine proteases was noted. The use of both tri- and tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl substitution of the urea moiety was beneficial, especially when strong electron-withdrawing groups where attached at the para position. Modest antiviral activity was found in a plaque reduction assay.

Potent beta-lactam inhibitors of human cytomegalovirus protease.

A series of novel monobactam inhibitors of human cytomegalovirus (HCMV) protease has been described that possess a heterocyclic thiomethyl side chain at C-4. Changes to the heterocycle did not significantly change the inhibitory activity of these compounds in an enzymatic assay, although improvements in solubility and cell culture activity were noted. A number of permutations between C-4 substitutions and N-1 derivatives led to the identification of several beta-lactams with antiviral activity in a plaque reduction assay. N-methyl thiotetrazole-containing compounds were found to be the most potent inhibitors in the enzymatic assay.

Comparison of industrial yeast strains for fermentation of spent sulphite pulping liquor fortified with wood hydrolysate.

Ethanol production from spent sulphite pulping liquor (SSL) was compared for four different yeasts. A second strain of S. cerevisiae as well as a 2-deoxyglucose-resistant strain formed through protoplast fusions between S. uvarum and S. diastaticus produced up to 27% more ethanol from SSL fortified with hydrolysis sugars than was produced by S. cerevisiae. The incremental improvement in ethanol yield appeared to vary with the degree of fortification, ranging from 5.8% for unfortified SSL, to 27% for the highest level of fortification tested. Decreasing fermentation rates were observed for SSL fortified with glucose, mannose and galactose, respectively. Sugar uptake rates in SSL fortified with glucose, galactose and mannose were 6.8, 2.8 and 2.0 g L-1 h-1, respectively. However, when these sugars were fermented along with a glucose cosubstrate, the rate at which the combined glucose/mannose medium was fermented was nearly identical to that of the glucose control.

Self-inflicted burns.

Self-inflicted burns are a regular source of admissions to burns units world wide. This study examines the characteristics and outcomes of those who deliberately burn themselves. The medical records of all patients admitted to the Royal Brisbane Hospital Burns Unit and identified as having suffered a self-inflicted burn between 1990 and 1995 were reviewed. The records of patients who doused themselves with flammable liquid between 1984 and 1995 were examined as a separate group. Of 1072 admissions there were 44 cases (4.1 per cent) of deliberately self-inflicted burns. Average age was 30 yr with an average total burn surface area (TBSA) of 30 per cent (range 1-98 per cent). Schizophrenia, depression and personality disorder were diagnosed in 71 per cent. Alcohol intoxication was common in the rest. Suicide attempters were almost all male and the majority (60 per cent) were diagnosed with a major psychiatric illness. Self-mutilators suffered much less serious burns and none died. Self-inflicted burns accounted for 24 per cent of burns admitted to the intensive care unit. Self-immolation with flammable liquid resulted in severe burns with a 45 per cent mortality. A number of differences was demonstrated between those patients who had attempted suicide and those who had deliberately burnt themselves without suicidal attempt. Self-immolators constitute a considerable proportion of major burns admitted to this unit.

A high containment polymodal pilot-plant fermenter--design concepts.

A 225 dm3 pilot-plant bioreactor system has been designed and constructed that is suitable for biohazardous fermentations. The design enables operation at containment levels above the requirements of good industrial large-scale practice (GILSP) without secondary containment of the whole plant. The main biosafety features of the systems include the use of steam barriers on O-ring seals, supply lines and stirrer seals, multiple O-ring seals, piping of condensate lines and pressure relief systems to a 'kill tank', double filtration of inlet and off gases and a mobile isolation unit that allows localised containment of sample valve and probe entry ports. The fermenter can, with minor modifications, be operated as a bottom-or top-stirred reactor for the culture of microbial or animal cells, or as an airlift reactor. The design offers considerable flexibility that could prove cost-effective for process development and production. The relevance of the various design features to enable bioreactor operations at pilot-plant scale to be carried out in compliance with current guidelines for large-scale culture of recombinant microorganisms and microbial pathogens is discussed.

The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.

The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.

A comparison of unit process systems for anchorage dependent cells.

The performance of three unit process systems for anchorage-dependent cells, namely the APV plate heat exchanger, glass bead reactor and microcarrier culture are reviewed. All three systems have been used for the production of various viruses and protein products and their use in a production process is related to various scaling-up parameters.