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Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages.

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.

Bacteria are becoming increasingly insensitive to antibiotics due to the spread of resistance. To take this challenge, the development of alternative therapies like bacteriophage therapy is urgently needed. Phages are viruses of bacteria that specifically recognize strains of a certain type of bacteria, penetrate and destroy them by cell lysis. Particularly in Eastern Europe, phages have been successfully used in the treatment of infections for decades. However, they have not yet been approved as drugs in the EU due to the lack of quality standards and clinical trials. The Fraunhofer ITEM is working on a platform-like manufacturing process for phages as active pharmaceutical ingredients with the aim of establishing them as drugs in the fight against bacterial infections in a wide range of indications and dosage forms.

Seit vielen Jahren wird Bacillus megaterium für die Produktion von rekombinanten Proteinen als Alternative zum etablierten Escherichia coli genutzt. Dafür wird bislang hauptsächlich das für B. megaterium optimierte Xylose-induzierbare Promotorsystem genutzt. Für die Regulation dieses Systems wird bislang angenommen, dass das Repressorprotein XylR in Abwesenheit von Xylose (reprimierter Zustand) an einen entsprechenden Operator an die DNA bindet und so die Transkription verhindert, während sich XylR bei Anwesenheit von Xylose (exprimierter Zustand) von der DNA löst und die Transkription ermöglicht. Im ersten Teil dieser Arbeit wurde der Repressor XylRB.meg erfolgreich gereinigt. Damit konnte durch DNA-Bindestudien in An- und Abwesenheit von Xylose erstmals gezeigt werden, dass beide Repressor-Formen (+/- Xylose) an den gleichen Operator binden. Allerdings werden beide Formen unterschiedlich oligomerisiert, sodass die DNA der Promotorregion in Abhängigkeit der gebundenen Repressor-Form unterschiedlich organisiert wird. Mit Hilfe von Footprint-Analysen wurde die Hypothese bestätigt, dass der Xylose-freie Repressor Loop-ähnliche DNA-Strukturen ausbildet, die zur Reprimierung der Transkription führen. Das bestehende Modell der XylR-vermittelten Regulation muss also entsprechend angepasst werden. Im zweiten Teil der Arbeit wurden mögliche Ursachen für die Ausbildung einer phänotypischen Heterogenität während der rekombinanten Proteinproduktion untersucht. Hierfür wurden verschiedene B. megaterium-Mutanten und B. megaterium mit Plasmiden unterschiedlichem Replikationsursprungs getestet. Dabei wurde Gfp als Modellprotein genutzt. Der Einfluss der Expression des Xylose-Transportergens, des Repressor-Induktor Verhältnis bei der Nutzung von Plasmiden hoher Kopienzahlen und die Verteilung von Zellkomponenten während der Zellteilung sollten so bestimmt werden. Durch Fluoreszenz-Durchflusszytometrie und Fluoreszenzmikroskopie Gfp-produzierender B. megaterium-Zellen wurde festgestellt, dass die phänotypische Heterogenität während der rekombinanten Gfp-Produktion im Zusammenhang mit einer ungleichen Plasmidverteilung steht. Dabei erhalten die Zellen mit älteren Zellpolen nach jeder Zellteilung mehr Plasmidkopien als die Zellen mit jüngeren Zellpolen, um durch sogenanntes „Bet-hedging“ Vorteile bei sich wechselnden Umweltbedingungen zu haben., Since many years, Bacillus megaterium is used for the production and secretion of recombinant proteins as an alternative host to the well-characterized Escherichia coli. For this purpose, an optimized xylose-inducible expression system was usually employed. It was postulated that in the absence of xylose the xylose repressor XylR binds to the operator and prevents expression of the following genes. In the presence of xylose XylR binds the sugar, which induces a structural rearrangement responsible for the loss of affinity of XylR for its DNA target and induction of gene expression. So far, no experimental evidence exists for this model, since the DNA-binding behaviour of XylR was not analysed. In the first part of this work the xylose repressor was recombinantly produced and successfully purified. In vitro DNA-binding studies showed that XylR with and also without bound xylose binds to the same operator OxylA. However, both forms of XylR (+/- Xylose) revealed different states of oligomerization, which resulted in a different structural organization of the bound operator DNA. Footprint analyses confirmed the hypothesis, that the xylose-free XylR forms DNA-loop structures causing repression of gene expression. The failure of the DNA bound xylose containing XylR to form these structure allows for gene expression. Based on all results, the postulated model of XylR-DNA binding needs to be adapted. Utilization of the plasmid-based xylose-inducible expression system leads to phenotypic heterogeneity during the recombinant protein production. Hence, in the second part of this work the molecular basis for phenotypic heterogeneity was analysed. For this purpose, B. megaterium mutants were tested with plasmids carrying different origins of replication. Gfp served as model protein. The influence of xylose transporter gene expression, imbalanced repressor-inducer equilibrium due to the utilization of high copy plasmids and unequal distribution of cell components during cell divison were analyzed. Gfp quantification via fluorescence flow cytometry and fluorescence microscopy demonstrated that phenotypic heterogeneity is due to an unequal distribution of plasmids during cell division. The cells harboring an older cell pole receive more plasmid copies after each cell division compared to the cells derived from the younger cell pole. It is assumed that bacteria use this so-called bet-hedging strategy to obtain advantages in a changing environments.