Isolierung und Charakterisierung eines phagenähnlichen Bacteriocins und eines virulenten Phagen und deren therapeutische Einsatzmöglichkeiten gegen Yersinia enterocolitica-Infektionen

Durch die wachsende Anzahl von multiresistenten Bakterien, die auch durch den Mißbrauch von Antibiotika als Masthilfsmittel in der Tierzucht entstanden sind, erlangen alternative Methoden zur Bekämpfung bakterieller Infektionen ihre Bedeutung zurück. Diese Arbeit befaßt sich mit zwei Substanzen, um Infektionen mit Yersinia (Y.) enterocolitica einzudämmen. Es wurde in dieser Arbeit ein Bacteriocin aus Y. enterocolitica isoliert und charakterisiert. Das Reinigungschema folgte den Strategien der Phagenaufreinigung, angeschlossen wurde zur Überprüfung der Reinheit ein Gelfiltrationsschritt. Die Eigenschaften des gereinigten Enterocoliticins wurden in vitro und in vivo getestet. Im Zellkulturversuch zeigte sich das Enterocoliticin in der Abtötung von an eukaryonte Zellen adhärierten Bakterien als sehr wirksam, in eukaryonte Zellen eingewanderte Bakterien wurden hingegen nicht abgetötet. Aufgrund dieser vielversprechenden Ergebnisse wurde der Therapieansatz im Mausmodell angewendet. Das Mausmodell ist für Y. enterocolitica ein bereits erprobtes Modell. Die Tiere wurden oral infiziert, um den natürlichen Infektionsweg nachzustellen, das Enterocoliticin wurde ebenfalls oral verabreicht. Die Infektion wurde durch die Enterocoliticingabe nur unwesentlich beeinflußt, auch gelang der Nachweis des Enterocoliticins weder im Gastrointestinaltrakt noch in den Faeces. Die Therapie der infizierten Mäuse gelang auf diese Weise nicht. Weiterhin wurde ein Yersinia-Phage aus Schweinegülle isoliert, gereinigt und charakterisiert. Es handelt sich um einen T4-ähnlichen, virulenten Phagen mit einer Genomgröße von ca. 50 kbp und einem weiten Wirtsspektrum in Yersinia, das sogar speziesübergreifend ist. Da der Phage bei 37°C die Wirtszelle lysiert und durch seine hohe Wirksamkeit in vitro erschien der Phage von seinen Eigenschaften her zur Phagentherapie als geeignet. Es wurden analog zum Enterocoliticin-Tierexperiment Mäuse mit Y. enterocolitica oral infiziert, diesen Tieren wurde der Phage auf unterschiedlichen Wegen und zu unterschiedlichen Zeitpunkten appliziert. Die Tiere zeigten bei der parenteralen Gabe keinerlei Unverträglichkeitserscheinungen, bei der oralen Gabe wurde der Magensaft zuvor abgepuffert. Der Therapieerfolg im Vergleich zur Kontrollgruppe war wenig vielversprechend, es zeigten sich sehr ähnliche Infektionsverläufe in Kontroll-und Therapiegruppe. Diese beiden untersuchten Alternativwege zur Behandlung von Yersiniosen erwiesen sich bei den angewendeten Methoden bislang als nicht erfolgreich, dennoch muß auf diesem Gebiet weitergeforscht werden, wie erfolgreiche Therapieansätze aus anderen Tiermodellen zeigen. Es wirken nur wenige Phagen im Organismus als Therapeutikum, dennoch müssen diese Untersuchungen unternommen werden, um im Organismus wirksame, antibakterielle Substanzen zu finden und um einen Alternativweg zur Antibiotikumtherapie zu entwickeln.The increasing number of multi-resistant bacteria, which resulted also from the abuse of antibiotics as mast additives in animal breeding, alternative methods regain importance for the combat at bacterial infections back. In this work two substances were investigated to restrict infections with Yersinia (Y.) enterocolitica. In this study a bacteriocin from Y. enterocolitica, designated enterocoliticin, was isolated and characterized. The purification strategy followed protocols of phage isolation. The purified preparations were examined by final gel filtration step. The properties of the purifed enterocoliticin were tested in vitro and in vivo. In a cell culture assay enterocoliticin was able to kill bacteria adherent to eukaryontic cells very effectively, however, bacteria invaded into eukaryotic cells were not affected. Due to these results enterocoliticin was applied in a mouse-infection-model in a therapeutic attempt. The mouse infection model is a well established system for infections with Y. enterocolitica. The animals were orally infected with Yersinia, and the enterocoliticin was orally applied, too. The infection was only insignificantly influenced by enterocoliticin. In addition of enterocoliticin was not detected succeeded in the gastro-intestinal-tract or in the faeces. The therapy of the infected mice did not succeed in this way. Furthermore, a Yersinia phage from pig manure was isolated and characterized. It is a T4 phage like virulent phage, containing a genom of approx. 50 kbp and posessing a wide host-range in Yersinia. Because of lytic properties of the phage at 37°C and his high effectiveness in vitro the phage appeared to be for phage therapy experiments. Similarly to the enterocoliticin experiment mice were infected with Y. enterocolitica orally, these animals were treated with the phage on different application routes and different time points. The animals did not show any incompatibilities upon parenteral gift. Before oral administration of the phage the gastric juice was buffered. Therapy outcome in comparison to the control group was little promising, it revealed a very similar infection process in control group and therapy group. These two investigated alternative ways for the control of Yersinia infections did not prove successful with the applied methods, however, further research must be carried out as successful therapeutical experiments from other animal models showed. It has to be considered that not all phages are appropriate as therapeutical agent however, more studies must be conducted to find more appropriate substances, which may work as effective antibacterial substances to develop alternative ways to antibiotic therapy

Cluster analysis of resistance combinations in Escherichia coli from different human and animal populations in Germany 2014-2017

Recent findings on Antibiotic Resistance (AR) have brought renewed attention to the comparison of data on AR from human and animal sectors. This is however a major challenge since the data is not harmonized. This study performs a comparative analysis of data on resistance combinations in Escherichia coli (E. coli) from different routine surveillance and monitoring systems for human and different animal populations in Germany. Data on E. coli isolates were collected between 2014 and 2017 from human clinical isolates, non-clinical animal isolates from food-producing animals and food, and clinical animal isolates from food-producing and companion animals from national routine surveillance and monitoring for AR in Germany. Sixteen possible resistance combinations to four antibiotics—ampicillin, cefotaxime, ciprofloxacin and gentamicin–for these populations were used for hierarchical clustering (Euclidian and average distance). All analyses were performed with the software R 3.5.1 (Rstudio 1.1.442). Data of 333,496 E. coli isolates and forty-one different human and animal populations were included in the cluster analysis. Three main clusters were detected. Within these three clusters, all human populations (intensive care unit (ICU), general ward and outpatient care) showed similar relative frequencies of the resistance combinations and clustered together. They demonstrated similarities with clinical isolates from different animal populations and most isolates from pigs from both non-clinical and clinical isolates. Isolates from healthy poultry demonstrated similarities in relative frequencies of resistance combinations and clustered together. However, they clustered separately from the human isolates. All isolates from different animal populations with low relative frequencies of resistance combinations clustered together. They also clustered separately from the human populations. Cluster analysis has been able to demonstrate the linkage among human isolates and isolates from various animal populations based on the resistance combinations. Further analyses based on these findings might support a better one-health approach for AR in Germany.Peer Reviewe

Proposal of Epidemiological Cutoff Values for Apramycin 15 μg and Florfenicol 30 μg Disks Applicable to Staphylococcus aureus

Funding Information This study was supported by Project BIOSAFE funded by FEDER through the Programa Operacional Factores de Competitividade–COMPETE and by Fundação para a Ciência e a Tecnologia (FCT, Portugal)—Grant LISBOA-01-0145-FEDER-030713, PTDC/CAL-EST/30713/2017 and by FCT through funds to GHTM (UID/04413/2020), CIISA Project (UID/CVT/00276/2020), and Project PTDC/CVT-CVT/28469/2017. The contributions of Andrea T. Feßler and Stefan Schwarz were financially supported by the German Federal Ministry of Education and Research (BMBF) under project numbers 01KI1727D and 01KI2009D as part of the Research Network Zoonotic Infectious Diseases. Part of this research was supported by Cost Action CA18217: European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT).Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.publishersversionpublishe

Towards a standardized method for broth microdilution susceptibility testing of Haemophilus parasuis

Currently, there is no agreed method available for broth microdilution susceptibility testing of Haemophilus parasuis, one of the most important bacterial pathogens in pig production. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. Growth determinations using one type strain and three field isolates revealed no visible growth of H. parasuis in media which have proven to be suitable for susceptibility testing of fastidious organisms. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. The reproducibility of MICs obtained in this medium was evaluated and statistically analyzed, considering a model with two different variables (precondition of five identical MICs and MIC mode accepting a deviation of ±1 dilution step, respectively). No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). Nearly all MICs of quality control strains were in the acceptable range. Subsequently, 47 H. parasuis isolates representing 13 serovars were tested with the newly developed medium and TMB. Statistical analysis of all isolates and 15 antimicrobial agents and antimicrobial combinations showed no significant difference between MICs obtained in supplemented CAMHB and TMB. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 24 h

Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids

Background: Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. Methods: In this study, Escherichia coli with the major ESC-R genes bla CTX-M-1, bla CTX-M-15, bla CTX-M-14 (ESBLs) and bla CMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n = 192 ESC-R E. coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. Findings: We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E. coli lineages and host species. The IncI1 pST3 bla CTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 bla CMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 bla CMY-2 plasmid in ST131 and ST117 E. coli harbouring this plasmid. The IncI1 pST2 bla CMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 bla CTX-M-15 and F2:A-:B- bla CTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 bla CTX-M-1 and IncF F31:A4:B1 bla CTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. Interpretation: We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. Funding: This project was supported by the (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain)

Defining the scope of the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet): a bottom-up and One Health approach

Background Building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) was proposed to strengthen the European One Health antimicrobial resistance (AMR) surveillance approach. Objectives To define the combinations of animal species/production types/age categories/bacterial species/specimens/antimicrobials to be monitored in EARS-Vet. Methods The EARS-Vet scope was defined by consensus between 26 European experts. Decisions were guided by a survey of the combinations that are relevant and feasible to monitor in diseased animals in 13 European countries (bottom-up approach). Experts also considered the One Health approach and the need for EARS-Vet to complement existing European AMR monitoring systems coordinated by the ECDC and the European Food Safety Authority (EFSA). Results EARS-Vet plans to monitor AMR in six animal species [cattle, swine, chickens (broilers and laying hens), turkeys, cats and dogs], for 11 bacterial species (Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus suis). Relevant antimicrobials for their treatment were selected (e.g. tetracyclines) and complemented with antimicrobials of more specific public health interest (e.g. carbapenems). Molecular data detecting the presence of ESBLs, AmpC cephalosporinases and methicillin resistance shall be collected too. Conclusions A preliminary EARS-Vet scope was defined, with the potential to fill important AMR monitoring gaps in the animal sector in Europe. It should be reviewed and expanded as the epidemiology of AMR changes, more countries participate and national monitoring capacities improve.Peer reviewe

Therapierelevante Antibiotikaresistenzen im One-Health-Kontext

„One Health“ bezeichnet ein Konzept, das die Gesundheit von Menschen, Tieren und der Umwelt miteinander verbindet. In Deutschland gibt es umfangreiche Daten zur Antibiotikaresistenz (AMR) und multiresistenten Erregern (MRE) in der Human- und Veterinärmedizin sowie aus Untersuchungen in verschiedenen Umweltkompartimenten (Boden, Wasser, Abwasser). Die Erhebung erfolgt nach unterschiedlichen Vorgaben und Standards, was den Vergleich von Daten erschwert. Ein Fokus auf humantherapeutisch wichtige AMR und MRE ist hilfreich, um eine gewisse Orientierung vorzugeben. Die meisten Daten liegen sektorübergreifend zu Methicillin-resistenten Staphylococcus aureus und multiresistenten Enterobacterales wie Escherichia coli und Klebsiella pneumoniae vor. Hier sind die Trends der Resistenzen heterogen. Der Einsatz von Antibiotika führt zur Selektion von MRE, was gut dokumentiert ist. Erfolge bei der Minimierung des Antibiotikaeinsatzes konnten in zurückliegenden Jahren für einzelne Sektoren dargestellt und z. T. mit Erfolgen in der Eindämmung von AMR und MRE korreliert werden (Rückgang MRSA in der Humanmedizin). Auch sektorspezifische Maßnahmen zur Senkung der Last durch MRE und AMR sind notwendig, da Resistenzprobleme nicht generell eine Verknüpfung mit anderen Sektoren aufweisen. Carbapenemresistenzen sind vor allem bei pathogenen Erregern vom Menschen nachweisbar. Colistinresistenzen kommen in verschiedenen Sektoren vor, zeigen aber dort jeweils verschiedene Mechanismen. Resistenzen gegen Reservesubstanzen wie Linezolid sind in Deutschland selten, sie zeigen aber einen konkreten One-Health-Bezug. Bestrebungen zur Harmonisierung von Methoden, z. B. im Bereich der antimikrobiellen Empfindlichkeitstestung und genombasierten Erreger- und AMR-Surveillance, sind ein wichtiger erster Schritt zu einer Vergleichbarkeit der verschiedenen Datenerhebungen.One Health refers to a concept that links human, animal, and environmental health. In Germany, there is extensive data on antibiotic resistance (AMR) and multidrug-resistant (micro)organisms (MDRO) in human and veterinary medicine, as well as from studies in various environmental compartments (soil, water, wastewater). All these activities are conducted according to different specifications and standards, which makes it difficult to compare data. A focus on AMR and MDRO of human therapeutic importance is helpful to provide some guidance. Most data are available across sectors on methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant Enterobacterales such as Escherichia coli and Klebsiella pneumoniae. Here, the trends of resistance are heterogeneous. Antibiotic use leads to MRE selection, which is well documented. Success in minimizing antibiotic use has also been demonstrated in recent years in several sectors and could be correlated with success in containing AMR and MDRO (e.g., decrease in MRSA in human medicine). Sector-specific measures to reduce the burden of MDRO and AMR are also necessary, as not all resistance problems are linked to other sectors. Carbapenem resistance is still rare, but most apparent in human pathogens. Colistin resistance occurs in different sectors but shows different mechanisms in each. Resistance to antibiotics of last resort such as linezolid is rare in Germany, but shows a specific One Health correlation. Efforts to harmonize methods, for example in the field of antimicrobial susceptibility testing and genome-based pathogen and AMR surveillance, are an important first step towards a better comparability of the different data collections.Peer Reviewe

Genetic landscape of pediatric acute liver failure of indeterminate origin.

BACKGROUND AIMS Pediatric acute liver failure (PALF) is a life-threatening condition. In Europe, main causes are viral infections (12-16%) and inherited metabolic diseases (14-28%). Yet, in up to 50% of cases the underlying etiology remains elusive, challenging clinical management, including liver transplantation. We systematically studied indeterminate PALF cases referred for genetic evaluation by whole-exome sequencing (WES), and analyzed phenotypic and biochemical markers, and the diagnostic yield of WES in this condition. METHODS With this international, multicenter observational study, patients (0-18 y) with indeterminate PALF were analyzed by WES. Data on the clinical and biochemical phenotype were retrieved and systematically analyzed. RESULTS In total, 260 indeterminate PALF patients from 19 countries were recruited between 2011 and 2022, of whom 59 had recurrent PALF (RALF). WES established a genetic diagnosis in 37% of cases (97/260). Diagnostic yield was highest in children with PALF in the first year of life (46%), and in children with RALF (64%). Thirty-six distinct disease genes were identified. Defects in NBAS (n=20), MPV17 (n=8) and DGUOK (n=7) were the most frequent findings. When categorizing, most frequent were mitochondrial diseases (45%), disorders of vesicular trafficking (28%) and cytosolic aminoacyl-tRNA synthetase deficiencies (10%). One-third of patients had a fatal outcome. Fifty-six patients received liver transplants. CONCLUSION This study elucidates a large contribution of genetic causes in PALF of indeterminate origin with an increasing spectrum of disease entities. The high proportion of diagnosed cases and potential treatment implications argue for exome or in future rapid genome sequencing in PALF diagnostics

Pilot testing the EARS-Vet surveillance network for antibiotic resistance in bacterial pathogens from animals in the EU/EEA

IntroductionAs part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis.MethodsEleven partners from nine EU/EEA countries participated and shared available data for the period 2016–2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations).ResultsCollected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria–antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species).DiscussionKey issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria–antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis