Antimicrobial activity of fosfomycin and tobramycin in combination against cystic fibrosis pathogens under aerobic and anaerobic conditions

There is a need for new antibiotics or combination of antibiotics that possess activity against increasingly resistant cystic fibrosis (CF) respiratory pathogens such as Pseudomonas aeruginosa and MRSA.

A PLATE ASSAY FOR THE DETECTION OF ORGANOPHOSPHONATE MINERALIZATION BY ENVIRONMENTAL BACTERIA, AND ITS MODIFICATION AS AN ACTIVITY STAIN FOR IDENTIFICATION OF THE CARBON-PHOSPHORUS BOND-CLEAVAGE ENZYME PHOSPHONOACETATE HYDROLASE

A replica plate screening technique, based on the acid molybdate assay for detection of phosphate has been developed to permit the detection of microorganisms capable of mineralizing organophosphonates. The method was further adapted as the basis of an activity stain for the detection of the carbon - phosphorus bond cleavage enzyme phosphonoacetate hydrolase in PAGE gels.

Alternative method for producing organic fertiliser from anaerobic digestion liquor and limestone powder:High Shear wet granulation

Generally, the solid and liquid fractions (digestate) from Anaerobic Digestion (AD) energy production are considered as waste. This has a negative impact on the sustainability of AD processes because of the financial outlay required to treat digestate before being discharged into municipal water treatment plants or natural water bodies. The main aim of this research was to investigate feasibility of producing an organic fertiliser using anaerobic digestate and limestone powders as the raw materials employing a high shear granulation process. Two-level factorial experimental design was used to determine the influence of granulation process variables on, the strength, resistance to attrition and yield of the granules. It was concluded from the study that it is technically feasible to produce organic fertiliser granules of acceptable strength and product yield. Increasing the liquid-to-solid ratio during granulation leads to increased granule strength and better product yield. Although the strength of the granules produced was lower than typical strength of commercial synthetic fertiliser granules (about 5 to 7. MPa), this could be improved by mixing the digestate with a polymeric binder or coating the particles post granulation. © 2012 Elsevier B.V.

The impact of antibiotic use on the incidence and resistance pattern of extended-spectrum beta-lactamase-producing bacteria in primary and secondary healthcare settings

Aims: The objective of the present study was to study the relationship between hospital antibiotic use, community antibiotic use and the incidence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in hospitals, while assessing the impact of a fluoroquinolone restriction policy on ESBL-producing bacteria incidence rates. METHODS: The study was retrospective and ecological in design. A multivariate autoregressive integrated moving average (ARIMA) model was built to relate antibiotic use to ESB-producing bacteria incidence rates and resistance patterns over a 5 year period (January 2005-December 2009). Results: Analysis showed that the hospital incidence of ESBLs had a positive relationship with the use of fluoroquinolones in the hospital (coefficient = 0.174, P= 0.02), amoxicillin-clavulanic acid in the community (coefficient = 1.03, P= 0.03) and mean co-morbidity scores for hospitalized patients (coefficient = 2.15, P= 0.03) with various time lags. The fluoroquinolone restriction policy was implemented successfully with the mean use of fluoroquinolones (mainly ciprofloxacin) being reduced from 133 to 17 defined daily doses (DDDs)/1000 bed days (P <0.001) and from 0.65 to 0.54 DDDs/1000 inhabitants/day (P= 0.0007), in both the hospital and its surrounding community, respectively. This was associated with an improved ciprofloxacin susceptibility in both settings [ciprofloxacin susceptibility being improved from 16% to 28% in the community (P <0.001)] and with a statistically significant reduction in ESBL-producing bacteria incidence rates. Discussion: This study supports the value of restricting the use of certain antimicrobial classes to control ESBL, and demonstrates the feasibility of reversing resistance patterns post successful antibiotic restriction. The study also highlights the potential value of the time-series analysis in designing efficient antibiotic stewardship. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.

Toxicity of mono-, di- and tri-chlorophenols to lux marked terrestrial bacteria, Burkholderia species Rasc c2 and Pseudomonas fluorescens

Burkholderia species RASC and Pseudomonas fluorescens were marked with lux genes, encoding for bioluminescence and used to assess the toxicity of mono-, di- and tri-chlorophenols by determining the decline in bioluminescence following exposure to the compounds in aqueous solution. Toxicity was expressed as a 50% effective concentration value (EC50, equating to the concentration of compound which caused a 50% decline in bioluminescence. Comparing the toxicity values of the compounds showed that, in general, increasing the degree of chlorination, increased toxicity. By carrying out forward multiple linear regressions with log10 EC50 values and physio-chemical descriptors, it was shown that molecular parameters describing the hydrogen bonding nature of a chlorophenol provided a better fit than regressions between toxicity data and log10 Kow alone. Utilising these descriptor variables in equations, it was shown that the toxicity of chlorophenols to the lux marked bacteria could be predicted from the compounds physio-chemical characteristics. By correlating lux marked RASC c2 and P. fluorescens EC50 values with toxicity values using Pimephales promelas (fathead minnow), Tetrahymena pyriformis (ciliate) and marine bacterium Vibriofischeri, it was apparent that lux marked RASC c2 correlated well with the freshwater aquatic species (P. promelas and T. pyriformis). This implied that for predictions of toxicity of organic xenobiotic compounds to higher organisms, lux marked RASC c2 could be utilised as a rapid surrogate.

Quantitative structure-toxicity relationships for chlorophenols to bioluminescent lux-marked bacteria using atom-based semi-empirical molecular-orbital descriptors

Literature data on the toxicity of chlorophenols for three luminescent bacteria (Vibrio fischeri, and the lux-marked Pseudomonas fluorescens 10586s pUCD607 and Burkholderia spp. RASC c2 (Tn4431)) have been analyzed in relation to a set of computed molecular physico-chemical properties. The quantitative structure-toxicity relationships of the compounds in each species showed marked differences when based upon semi-empirical molecular-orbital molecular and atom based properties. For mono-, di- and tri-chlorophenols multiple linear regression analysis of V. fischeri toxicity showed a good correlation with the solvent accessible surface area and the charge on the oxygen atom. This correlation successfully predicted the toxicity of the heavily chlorinated phenols, suggesting in V. fischeri only one overall mechanism is present for all chlorophenols. Good correlations were also found for RASC c2 with molecular properties, such as the surface area and the nucleophilic super-delocalizability of the oxygen. In contrast the best QSTR for P. fluorescens contained the 2nd order connectivity index and ELUMO suggesting a different, more reactive mechanism. Cross-species correlations were examined, and between V. fischeri and RASC c2 the inclusion of the minimum value of the nucleophilic susceptibility on the ring carbons produced good results. Poorer correlations were found with P. fluorescens highlighting the relative similarity of V. fischeri and RASC c2, in contrast to that of P. fluorescens.

Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection

The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10(8)-10(9) c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10(8)-10(9) c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of intrastrain antigenic variation of Bacteroides fragilis surface polysaccharides by monoclonal antibody labelling

Bacteroides fragilis is a constituent of the normal resident microbiota of the human intestine and is the gram-negative obligately anaerobic bacterium most frequently isolated from clinical infection. Surface polysaccharides are implicated as potential virulence determinants. We present evidence of within strain immunochemical variation of surface polysaccharides in populations that are noncapsulate by light microscopy as determined by monoclonal antibody labelling. Expression of individual epitopes can be enriched from a population of an individual strain by use of immunomagnetic beads. Also, individual colonies in which either >94% or 94% of the bacteria carry a given epitope, there is no enrichment for other epitopes recognized by different polysaccharide-specific monoclonal antibodies. This intrastrain variation has important implications for the development of potential vaccines or immunodiagnostic tests.

Lung Microbiota and Bacterial Abundance in Patients with Bronchiectasis when Clinically Stable and during Exacerbation

RATIONALE: Characterization of bacterial populations in infectious respiratory diseases will provide improved understanding of the relationship between the lung microbiota, disease pathogenesis and treatment outcomes.

OBJECTIVES: To comprehensively define lung microbiota composition during stable disease and exacerbation in bronchiectasis patients.

METHODS: Sputum was collected from patients when clinically stable and before and after completion of antibiotic treatment of exacerbations. Bacterial abundance and community composition were analyzed using anaerobic culture and 16S rDNA pyrosequencing.

MEASUREMENTS AND MAIN RESULTS: In clinically stable patients, aerobic and anaerobic bacteria were detected in 40/40 (100%) and 33/40 (83%) sputum samples, respectively. The dominant organisms cultured were P. aeruginosa (n=10 patients), H. influenzae (n=12), Prevotella (n=18) and Veillonella (n=13). Pyrosequencing generated over 150,000 sequences, representing 113 distinct microbial taxa; the majority of observed community richness resulted from taxa present in low abundance with similar patterns of phyla distribution in clinically stable patients and patients at the onset of exacerbation. Following treatment of exacerbation, there was no change in total (p=0.925), aerobic (p=0.917) or anaerobic (p=0.683) load and only a limited shift in community composition. Agreement for detection of bacteria by culture and pyrosequencing was good for aerobic bacteria such as P. aeruginosa (kappa=0.84) but poorer for other genera including anaerobes. Lack of agreement was largely due to bacteria been detected by pyrosequencing but not by culture.

CONCLUSIONS: A complex microbiota is present in the lungs of bronchiectasis patients which remains stable through treatment of exacerbations suggesting that changes in microbiota composition do not account for exacerbations.

Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Background: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).

Methodology/Principal Findings: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF Delta ARE and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells.

Conclusions/Significance: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.

Inflammatory bowel disease, gut bacteria and probiotic therapy

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD) and both diseases lead to high morbidity and health care costs. Complex interactions between the immune system, enteric commensal bacteria and host genotype are thought to underlie the development of IBD although the precise aetiology of this group of diseases is still unknown. The understanding of the composition and complexity of the normal gut microbiota has been greatly aided by the use of molecular methods and is likely to be further increased with the advent of metagenomics and metatranscriptomics approaches, which will allow an increasingly more holistic assessment of the microbiome with respect to both diversity and function of the commensal gut microbiota. Studies thus far have shown that the intestinal microbiota drives the development of the gut immune system and can induce immune homeostasis as well as contribute to the development of IBD. Probiotics which deliver some of the beneficial immunomodulatory effects of the commensal gut microbiota and induce immune homeostasis have been proposed as a suitable treatment for mild to moderate IBD. This review provides an overview over the current understanding of the commensal gut microbiota, its interactions with the mucosal immune system and its capacity to induce both gut homeostasis as well as dysregulation of the immune system. Bacterial-host events, including interactions with pattern recognition receptors (PRRs) expressed on epithelial cells and dendritic cells (DCs) and the resultant impact on immune responses at mucosal surfaces will be discussed. (C) 2009 Elsevier GmbH. All rights reserved.

Therapeutic Effects of Human Mesenchymal Stem Cells in Ex Vivo Human Lungs Injured with Live Bacteria

Rationale: Mesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells' immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury. Objectives: Therefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance. Methods: We developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury. Measurements and Main Results: Clinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria. Conclusions: In E. coli-injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.

Quinolones sensitize gram-negative bacteria to antimicrobial peptides

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. Antimicrobial peptides (APs) make up the front line of defense in those areas exposed to microorganisms, and there is intensive research to explore their use as new antibacterial agents. On the other hand, it is known that subinhibitory concentrations of antibiotics affect the expression of numerous bacterial traits. In this work we evaluated whether treatment of bacteria with subinhibitory concentrations of quinolones may alter the sensitivity to APs. A 1-h treatment of Klebsiella pneumoniae with 0.25 x the MIC of ciprofloxacin rendered bacteria more sensitive to polymyxins B and E, human neutrophil defensin 1, and beta-defensin 1. Levofloxacin and nalidixic acid at 0.25 x the MICs also increased the sensitivity of K. pneumoniae to polymyxin B, whereas gentamicin and ceftazidime at 0.25 x the MICs did not have such an effect. Ciprofloxacin also increased the sensitivities of K. pneumoniae ciprofloxacin-resistant strains to polymyxin B. Two other pathogens, Pseudomonas aeruginosa and Haemophilus influenzae, also became more sensitive to polymyxins B and E after treatment with 0.25 x the MIC of ciprofloxacin. Incubation with ciprofloxacin did not alter the expression of the K. pneumoniae loci involved in resistance to APs. A 1-N-phenyl-naphthylamine assay showed that ciprofloxacin and levofloxacin increased the permeabilities of the K. pneumoniae and P. aeruginosa outer membranes, while divalent cations antagonized this action. Finally, we demonstrated that ciprofloxacin and levofloxacin increased the binding of APs to the outer membrane by using dansylated polymyxin B.