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Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

Background: Global spread of multidrug resistant (MDR) bacteria is one of the biggest threats in modern medicine. Acinetobacter baumannii is a feared cause of lung infections particularly in critically ill patients lacking antibiotic options. Therefore, rediscovery of phage therapy has gained attention as treatment option due to its specificity and effectivity. Aims: This preclinical study evaluated the efficacy and safety of a novel phage preparation in a mouse model of A. baumannii lung infection. Methods: Mice were infected with a MDR A. baumannii strain [1]. Phage Acibel004 [2] was produced as suspension including efficient depletion of endotoxins using a newly established protocol. Solvent or phages were sprayed intratracheally 12 hours after infection. Clinical parameters, bacterial load, cell influx, lung permeability and cytokine release were quantified and histopathological examination was performed. Results: Phage-treated mice recovered faster from infection-associated hypothermia. Phage treatment reduced bacterial loads in lungs and BAL 48 hours after infection. Moreover, lung permeability and cytokine release were reduced. Histopathological examination of lungs revealed lesser extent of inflammation in phage-treated mice with unaffected cellular recruitment. No adverse effects were observed. Conclusion: For the first time, a highly purified phage against A. baumannii was successfully used to treat lung infection in vivo. The current preclinical data support the concept of phage-based therapy against pulmonary A. baumannii infections. [1] Knapp, S. et al. Am J Respir Crit Care Med 2006; 1;173(1):122-9 [2] Merabishvili, M. et al. PLoS One 2014; 11;9(8):e104853

Introduction: Chronic lung diseases are frequently complicated by lung and airway infections. The rise of new resistance mechanisms is alarming, and MDR bacteria, including Acinetobacter baumannii, are spreading globally. Rediscovery of phage-therapy could be a solution to overcome this problem. High specificity of bacteriophages and their effectivity in lysis make phages attractive as antibiotics are failing more and more. However, phages need to be properly chosen and characterized according to quality standards, and preparations must be highly purified.Objectives:This preclinical study aimed at determining the efficacy, safety and tolerability of a novel phage preparation in anticipation of a future clinical trial applying aerosolized lytic phages against A. baumannii. Methods: The specific phage Acibel004 [1], a myovirus, was produced as high-titer suspension subjected to final processing including highly efficient depletion of endotoxins by chromatography. Mice were transnasally infected with a MDR A. baumannii strain [2]. 12h p.i. mice were treated intratracheally with phage or solvent. At defined time points clinical parameters were measured, bacterial burdens in different organs determined and cell-influx analyzed. Lung permeability and cytokine release were measured and histological analyses performed. Furthermore, presence of vital phages was determined. Results: Phage application in uninfected mice did not cause measurable immune responses. In A. baumannii infected mice, phage therapy enhanced recovery from infection-associated hypothermia and improved clinical outcome. Bacterial loads in lungs and BALF of the phage-treated group were significantly reduced by 48h p.i.. Cellular recruitment to the lung and blood was unaffected by phage treatment, while lung permeability and cytokine production were significantly reduced. Hematoxylin and eosin stained lung slides of phage treated mice showed less spreading of bacteria to periphery. Phages were detected in BALF, lungs and plasma of treated mice. No adverse effects of phage therapy were observed. Conclusion: For the first time a highly purified phage preparation against A. baumannii was used successfully in vivo. The current preclinical data further support the concept of developing a phage-based therapy against pulmonary A. baumannii infections in patients.

Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a ?-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4) )(2) SO(4) , and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.

The inhibition potentials of products and substrate on the growth ofClostridium butyricum and Klebsiella pneumoniae in the glycerol fermentation are examined from experimental data and with a mathematicalmodel. Whereas the inhibition potential of externally added and self-produced 1,3-propanediol is essentially the same, butyric acid produced by the culture is more toxic than that externally added. The same seems to apply for acetic acid. The inhibitory effect of butyric acid is due tothe total concentration instead of its undissociated form. For acetic acid, it cannot be distinguished between the total concentration and the undissociated formThe inhibition effects of products and substrate in the glycerol fermentation are irrespective of the strains, and, therefore, the same growth model can be used. The maximum product concentrations tolerated (critical concentrations C(*) (pi)) are 0.35 g/Lfor undissociated acetic acid, 10.1 g/L for total butyric acid, 16.6 g/L for ethanol, 71.4 g/L for 1,3-propanediol, and 187.6 g/L for glycerol, which are applicable to C. butyricum and K. pneumoniae grown under a variety of conditions. For 55 steady-states, which were obtained from different types of continuous cultures over a pHrange of 5.3-8.5 and under both substrate limitation and substrate excess, the proposed growth model fits the experimental data with an average deviation of 17.0%. The deviation of model description from experimental values reduces of 11.4% if only the steady-states with excessive substrate are considered. (c) 1994 John Wiley & Sons, Inc.

Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.

Tryparedoxins (TXNs) are trypanothione-dependent peroxiredoxin oxidoreductases involved in hydroperoxide detoxification that have been shown to determine virulence in trypanosomatids. The structure of (15)N,(13)C-doubly-labeled, C-terminally-His-tagged tryparedoxin 1 from Crithidia fasciculata (Cf TXN1) was elucidated by three-dimensional NMR spectroscopy. Global folding was found to be similar to the crystal structure, but regions near the active site, especially the onset of helix alpha1 with the redox-active Cys 43 and helix alpha2 relevant to substrate binding, were less well defined in solution. The redox-inactive inhibitory substrate analogue N(1),N(8)-bis(ophthalmyl)spermidine was used to study the substrate/TXN interaction by two-dimensional (1)H,(15)N NMR spectroscopy. The NMR data complemented by molecular modeling revealed several alternative modes of ligand binding. The results confirm and extend the concept of TXN action and specificity derived from X-ray analysis and site-directed mutagenesis and thus improve the rational basis for inhibitor design.

The cultivation of genetically modified microorganisms, e.g.E. coli, larger than 100 g/L dry weight, requires fed-batch processing with aid of a controlled specific growth rate fi. A theoretical analysis of this kind of fed-batch processes, as well as a method to control /z will be discussed and the implementation of both in an industrial process control system will be shortly explained. The test of the control equipment is carried out with the help of on-line simulation methods. The experimental verification of the developed control methods is demonstrated with a high cell densityE. colicultivation.

A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h−1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h−1 all of these properties change due to derepression (for D 0.025 h−1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.

The exact time course of phosphate consumption in a tetracycline production byStreptomyces aureofaciens has been determined. The data have been compared with model simulations according to a model proposed by Votruba et al. (1984). This led to a revision of his equation for the rate of phosphate consumption and to the proposal that phosphate is consumed proportionally to the growth rate. In contradiction to the model simulations it was found that the length of the time lag of the production is independent of the initial phosphate concentration. While the model explains the time lag through inhibition of the production by phosphate, the measured data show that there must be another or an additional reason for the lag. Simultaneously with the start of the production the organism changes from an organic substrate to ammonia as nitrogen source.

A method based on the elemental balance of the process has been developed to calculate the consumption of cellulose in cellulase production byTrichoderma reesei Rut-C30. Using a synthetic medium only the oxygen in effluent gas and consumption of base for pH-correction has to be measured.