Total Synthesis of the GRP78-Downregulatory Macrolide (+)-Prunustatin A, the Immunosuppressant (+)-SW-163A, and a JBIR- 04 Diastereoisomer That Confirms JBIR-04 Has Nonidentical Stereochemistry to (+)-Prunustatin A

A unified total synthesis of the GRP78-downregulator(+)-prunustatin A and the immunosuppressant(+)-SW-163A based upon [1 + 1 + 1 + 1]-fragment condensationand macrolactonization between O(4) and C(5) is hereindescribed. Sharpless asymmetric dihydroxylation was used toset the C(2) stereocenter present in both targets. In like fashion,coupling of the (+)-prunustatin A macrolide amine with benzoicacid furnished a JBIR-04 diastereoisomer whose NMR spectradid not match those of JBIR-04, thus confirming that it hasdifferent stereochemistry than (+)-prunustatin A.

Inactivation of Mycobacterium avium subsp. paratuberculosis in milk during commercial pasteurisation

Four studies have been published relating to the inactivation of Mycobacterium avium subsp. paratuberculosis (Map) by commercial HTST pasteurization. Three of these were large surveys of commercially pasteurized milk at processing/retail level in the UK and Ontario, Canada, and the fourth a pasteurization study involving naturally infected milk and commercial-scale pasteurizing plant. Evidence that Map is capable of surviving commercial pasteurization was obtained in two of the studies: viable Map was cultured from 50 ml aliquots of commercially pasteurized milk after decontamination with 0.75% cetylpyridinium chloride for 5 h and then culture on Herrold's egg-yolk medium without antibiotics. In both studies culture did not commence until 24-72 h post-pasteurization and samples were stored at 4 degrees C in the interim period. In the other two milk surveys smaller volumes of milk were tested (1-5 ml and 15 ml) and no firm evidence of surviving Map was obtained. The three milk surveys differed in other respects - chemical decontamination, culture media used and use of antibiotics. Recent findings suggest that sub-lethally heat-injured Map in pasteurized milk have the potential to recover viability if stored at 4 degrees C for 48 h between heating and testing.

Decision making in childhood asthma exacerbation – a clinical judgement analysis

Background: Clinical decisions which impact directly on patient safety and quality of care are made during acute asthma attacks by individual doctors on the basis of their knowledge and experience. These include administration of systemic corticosteroids (CS), oral antibiotics, and admission to hospital. Clinical judgement analysis provides a methodology for comparing decisions between practitioners with different training and experience, and improving decision making. Methods: Stepwise linear regression was used to select clinical cues based on visual analogue scale assessments of the propensity of 62 clinicians to prescribe a short course of oral CS (decision 1), a course of antibiotics (decision 2), and/or admit to hospital (decision 3) for 60 â??paperâ?? patients. Results:When compared by specialty, paediatriciansâ?? models for decision 1 were more likely to include as a cue level of alertness (54% v. 16%); for decision 2 presence of crepitations (49% v. 16%), and less likely to include inhaled CS (8% v. 40%), respiratory rate (0% v. 24%), and air entry (70% v. 100%). When compared to other grades, the models derived for decision 3 by consultants/general practitioners were more likely to include wheeze severity as a cue (39% v. 6%). Conclusions: Clinicians differed in their use of individual cues and the number included in their models. Patient safety and quality of care will benefit from clarification of decision making strategies as general learning points during medical training, in the development of guidelines and care pathways, and by clinicians developing self-awareness of their own preferences.

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Cho SH, Naber K, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.

Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis.

Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

Chromosomal rearrangements affecting biofilm production and antibiotic resistance in a Staphylococcus epidermidis strain causing shunt-associated ventriculitis.

Ziebuhr W, Dietrich K, Trautmann M, Wilhelm M. Institut für Molekulare Infektionsbiologie, Würzburg, Germany. w.ziebuhr@mail.uni-wuerzburg.de During two clinical courses of shunt-associated meningitis in a 3-month-old child, five multiresistant S. epidermidis isolates were obtained and analyzed with regard to biofilm production and antibiotic susceptibility. Three S. epidermidis strains, which were initially isolated from the cerebrospinal fluid, produced biofilms on polystyrene tissue culture plates. Following antibiotic treatment and subsequent exchange of the shunt system, sterilization of the CSF was achieved. However, after three weeks a relapse of the infection occurred. The two S. epidermidis isolates obtained now were biofilm negative, but showed an identical resistance pattern as those from the previous infection, except that resistance to rifampicin and increased mininal inhibitory concentrations of aminoglycoside antibiotics had emerged. DNA fingerprinting by PFGE indicated the clonal origin of all isolates. However, some DNA rearrangements and differences in the IS256-specific hybridization patterns could be identified in the isolates from the second infection period that led to altered biofilm formation and increased expression of aminoglycoside resistance traits. The data evidence that variation of biofilm expression occurs in vivo during an infection and highlight the extraordinary genome flexibility of pathogenic S. epidermidis.

Comparative evaluation of 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide(XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the 1092 dilution MIC results revealed overall agreement, within the accuracy limits of the standard test ( 1 1092 dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test. (c) 2007 Elsevier B.V. All rights reserved.

Detection of Anaerobic Bacteria in High Numbers in Sputum from Patients with Cystic Fibrosis

Rationale: Pulmonary infection in cystic ?brosis (CF) is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway
mucus.
Objectives: To determine whether anaerobic bacteria are present in the sputum of patients with CF.
Methods: Sputum samples were collected from clinically stable adults with CF and bronchoalveolar lavage ?uid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriologic techniques and bacteria within the samples were quanti?ed and identi?ed.
Measurements and Main Results: Anaerobic species primarily within the genera Prevotella,Veillonella, Propionibacterium, andActinomyces were isolated in high numbers from 42 of 66 (64%) sputum samples from adult patients with CF. Colonization with Pseudomonas aeruginosa signi?cantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identi?ed in BALF from pediatric patients with CF. Although anaerobes were detected in induced sputum samples from 16 of 20 volunteers, they were present in much lower numbers and were
generally different species compared with those detected in CF sputum. Species-dependent differences in the susceptibility of the anaerobes to antibiotics with known activity against anaerobes were apparent with all isolates susceptible to meropenem.
Conclusions: A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing signi?cantly to infection and in?ammation in the CF
lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

The effect of treatment of cystic fibrosis pulmonary exacerbations on airways and systemic inflammation

Background: Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF Methods: Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out. Results: There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1). Conclusion: These data suggest a modifiable systemic response to IV antibiotics but a local sustained inflammatory response in the lung.

Quality of life and inflammation in exacerbations of bronchiectasis.

Patients with bronchiectasis often have impaired quality of life (QoL), which deteriorates with exacerbations. The aim of this study was to investigate changes in QoL and how these were influenced by changes in airway physiology and inflammation in patients with bronchiectasis before and after resolution of an exacerbation. Sputum induction and a QoL questionnaire were undertaken on the first day, day 14, and 4 weeks after completion of intravenous antibiotics (day 42). Eighteen patients (12 female) were recruited, median (IQ range) age of 54 (47–60) years. There was a trend towards an improvement in lung function from visit 1 to visit 2, but this was not statistically significant. C-reactive protein (CRP) [mean (SEM)] reduced between visit 1 and visit 2 [55.4 (21.5) vs 9.4 (3.1) mg/L, P = 0.03] but did not increase significantly on visit 3 [44.4 (32.9) mg/L, P = 0.27]. The median (interquartile range) sputum cell count (×106 cells/g of sputum) decreased from visit 1 to visit 2 [21.6 (11.8–37.6)–13.3 (6.7–22.9) × 106 cells/g, respectively, P = 0.008] and increased from visit 2 to visit 3 [26.3 (14.1–33.6) × 106 cells/g, P = 0.03]. All soluble markers of inflammation significantly reduced from visit 1 to visit 2 but increased on visit 3 with the exception of TNF-a. Regarding QoL, three of the four domains (dyspnoea, emotional, mastery) significantly improved from visit 1 to visit 2 but did not change between visit 2 and visit 3. The improvements in QoL scores could not be explained by the improvements in lung function or inflammatory markers.

Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations

Organic solvents are widely used in a range of multiphase bioprocess operations including the liquid-liquid extraction of antibiotics and two-phase biotransformation reactions. There are, however, considerable problems associated with the safe handling of these solvents which relate to their toxic and flammable nature. In this work we have shown for the first time that room-temperature ionic liquids, such as 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF6], can be successfully used in place of conventional solvents for the liquid-liquid extraction of erythromycin-A and for the Rhodococcus R312 catalyzed biotransformation of 1,3-dicyanobenzene (1,3-DCB) in a liquid-liquid, two-phase system. Extraction of erythromycin with either butyl acetate or [bmim][PF6] showed that values of the equilibrium partition coefficient, K, up to 20-25 could be obtained for both extractants. The variation of K with the extraction pH was also similar in the pH range 5-9 though differed significantly at higher pH values. Biotransformation of 1,3-DCB in both water-toluene and water-[bmim][PF6] systems showed similar profiles for the conversion of 1,3-DCB initially to 3-cyanobenzamide and then 3-cyanobenzoic acid. The initial rate of 3-cyanobenzamide production in the water-[bmim][PF6] system was somewhat lower, however, due to the reduced rate of 1,3-DCB mass transfer from the more viscous [bmim] [PF,] phase. it was also shown that the specific activity of the biocatalyst in the water-[bmim][PF6] system was almost an order of magnitude greater than in the water-toluene system which suggests that the rate of 3-cyanobenzamide production was limited by substrate mass transfer rather than the activity of the biocatalyst. (C) 2000 John Wiley & Sons, Inc.

Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis

This study compares conventional and molecular techniques for the detection of fungi in 77 adult cystic fibrosis (CF) patients. Three different methods were investigated, i.e., (1) conventional microbiological culture (including yeasts and filamentous fungi), (2) mycological culture with CF-derived fungal specific culture media, and (3) Non-culture and direct DNA extraction from patient sputa. Fungi isolated from environmental air samples of the CF unit were compared to fungi in sputa from CF patients. Fungi (n = 107) were detected in 14/77(18%) of patients by method 1, in 60/77 (78%) of patients by method 2 and with method 3, in 77/77(100%) of the patients. The majority of yeasts isolated were Candida albicans and C. dubliniensis. Exophiala (Wangiella) dermatitidis, Scedosporiumapiospermum, Penicillium spp., Aspergillus fumigatus, and Aspergillus versicolor were also identified by sequence analysis of the rDNA short internal transcribed spacer (ITS2) region. Conventional laboratory analysis failed to detect fungi in 63 patients mainly due to overgrowth by Gram-negative organisms. Mycological culture with antibiotics dramatically increased the number of fungi that could be detected. Molecular techniques detected fungi such as Saccharomyces cerevisiae, Malassezia spp., Fuscoporia ferrea, Fusarium culmorum, Acremonium strictum, Thanatephorus cucumeris and Cladosporium spp. which were not found with other methods. This study demonstrates that several potentially important fungi may not be detected if mycological culture methods alone are used. A polyphasic approach employing both enhanced mycological culture with molecular detection will help determine the presence of fungi in the sputa of patients with CF and their healthcare environment.

Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF)

Yeasts and filamentous fungi are beginning to emerge as significant microbial pathogens in patients with cystic fibrosis (CF), particularly in relation to allergic-type responses, as seen in patients with allergic bronchopulmonary aspergillosis (ABPA), Aspergillus bronchitis and in invasive fungal disease in lung transplant patients. Four fungal media were compared in this study, including Sabouraud Dextrose Agar (SDA) and Medium B, with and without the addition of selective antibiotics, where antibiotic-supplemented media were designated with (+). These media were compared for their ability to suppress contaminating, mainly Gram-ve pathogens, in CF sputa (Pseudomonas aeruginosa, Burkholderia cepacia complex [BCC] organisms) and to enhance the growth of fungi present in CF sputum. Medium B consisted of glucose (16.7 g/l), agar (20 g/l), yeast extract (30 g/l) and peptone (6.8 g/l) at pH 6.3 and both SDA(+) and Medium B+ were supplemented with cotrimethoxazole, 128 mg/l; chloramphenicol, 50 mg/l; ceftazidime, 32 mg/l; colistin, 24 mg/l). Employment of SDA(+) or Medium B+ allowed an increase in specificity in the detection of yeasts and moulds, by 42.8% and 39.3%, respectively, over SDA when used solely. SDA(+) had a greater ability than Medium B+ to suppress bacterial growth from predominantly Gram-ve co-colonisers. This is a significant benefit when attempting to detect and isolate fungi from the sputum of CF patients, as it largely suppressed any bacterial growth, with the exception of the BCC organisms, thus allowing for an increased opportunity to detect target fungal organisms in sputum and represented a significant improvement over the commercial medium (SDA), which is currently used. Overall, both novel selective media were superior in their ability to suppress bacteria in comparison with the commercially available SDA medium, which is routinely employed in most clinical microbiology diagnostic laboratories presently. Alternatively, Medium B+ had a great ability to grow fungi than SDA(+) and when employed together, the specificity of combined use was 82%, with a sensitivity for yeasts, filamentous fungi, and combined overall fungi of 96.0%, 92.3% and 96.0%, respectively. Overall, when employing one fungal selective medium for the routine detection of yeasts and filamentous fungi in the sputum of CF patients, we would recommend employment of Medium B+. However, we would recommend the combined employment of SDA(+) and Medium B+, in order to synergistically isolate and detect the greatest number of fungi present in CF sputa. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V All rights reserved.

Modelling the impact of antibiotic use and infection control practices on the incidence of hospital-acquired methicillin-resistant Staphylococcus aureus: a time-series analysis

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. A wide range of factors have been suggested to influence the spread of MRSA. The objective of this study was to evaluate the effect of antimicrobial drug use and infection control practices on nosocomial MRSA incidence in a 426-bed general teaching hospital in Northern Ireland.

Methods: The present research involved the retrospective collection of monthly data on the usage of antibiotics and on infection control practices within the hospital over a 5 year period (January 2000–December 2004). A multivariate ARIMA (time-series analysis) model was built to relate MRSA incidence with antibiotic use and infection control practices.

Results: Analysis of the 5 year data set showed that temporal variations in MRSA incidence followed temporal variations in the use of fluoroquinolones, third-generation cephalosporins, macrolides and amoxicillin/clavulanic acid (coefficients = 0.005, 0.03, 0.002 and 0.003, respectively, with various time lags). Temporal relationships were also observed between MRSA incidence and infection control practices, i.e. the number of patients actively screened for MRSA (coefficient = -0.007), the use of alcohol-impregnated wipes (coefficient = -0.0003) and the bulk orders of alcohol-based handrub (coefficients = -0.04 and -0.08), with increased infection control activity being associated with decreased MRSA incidence, and between MRSA incidence and the number of new patients admitted with MRSA (coefficient = 0.22). The model explained 78.4% of the variance in the monthly incidence of MRSA.

Conclusions: The results of this study confirm the value of infection control policies as well as suggest the usefulness of restricting the use of certain antimicrobial classes to control MRSA.

Effect of sub-lethal challenge with Photodynamic Antimicrobial Chemotherapy (PACT) on the antibiotic susceptibility of clinical bacterial isolates

This study aimed to determine the effect of sub-lethal challenge with Photodynamic Antimicrobial Chemotherapy (PACT) on the susceptibility of clinical Staphylococcus aureus and Pseudomonas aeruginosa isolates to both PACT and a range of antibiotics used in the treatment of infection caused by these bacteria. Clinical S. aureus and P. aeruginosa isolates were exposed to sub-lethal PACT with meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) and methylene blue (MB) over a 72 h period. After exposure, susceptibility of surviving organisms to a range of antibiotics was determined and compared with the susceptibility of an untreated control. Surviving bacteria were also exposed to previously lethal photosensitizer-light combinations, to determine if susceptibility to PACT was affected by sub-lethal exposure. Exposure to sub-lethal PACT did not decrease susceptibility to antibiotics with the minimum inhibitory concentrations for 95% and 100% of P. aeruginosa and S. aureus isolates, respectively, within two doubling dilutions of the MIC of the untreated control. Similarly, habituation with sub-lethal PACT did not reduce susceptibility of P. aeruginosa isolates to PACT levels previously determined as lethal. A reduction in susceptibility to PACT following habituation was apparent for two S. aureus isolates with MB and for 1 S. aureus isolate with IMP. However, for two of these three isolates, the log reduction for habituated cells was still greater than 4 log(10). PACT remains an attractive potential treatment for infection caused by these bacteria. (C) 2010 Elsevier B.V. All rights reserved.