ESBL-plasmids carrying toxin-antitoxin systems can be “cured” of wild-type Escherichia coli using a heat technique

Plasmid-encoded extended-spectrum beta-lactamase (ESBL)-enzymes are frequently produced by Escherichia coli. Several ESBL-plasmids contain genes for toxin- antitoxin (TA) systems, which assure the maintenance of plasmids in bacteria and prevent the cells from "post-segregational killing". These systems limit options to "cure" plasmids of ESBL-wild-type strains due to the death of the bacterial cells. A helpful tool to understand the role of ESBL-plasmids in the dissemination of pandemic multi-resistant E. coli are ESBL- plasmid-"cured"-variants (PCVs) and their comparison to ESBL-wild-type strains. The purpose of this study was to construct PCVs of ESBL-wild-type E. coli strains despite the presence of genes for TA systems. Using enhanced temperatures and brain-heart-infusion broth it was possible to construct viable PCVs of wild-type ESBL-E. coli strains. The occurrence of TA system- genes including hok/sok, srnB/C, vagC/D, pemI/K on ESBL-plasmids of replicon types FIA or FIB was demonstrated by bioinformatic analyses. The loss of the plasmid and the genetic identity of PCV and corresponding wild-type strain was confirmed via different methods including plasmid-profile-analysis, pulsed- field gel electrophoresis and bioinformatics using generated whole genome data of the strains. This short report describes the successful construction of viable PCVs of ESBL-wild-type E. coli strains. The results are hence surprising due to the fact that all "cured" ESBL-plasmids contained at least one complete toxin-antitoxin system, whose loss would normally mean the death of bacterial cells

Inactivation of airborne bacteria by plasma treatment and ionic wind for indoor air cleaning

Airborne bacteria are a general problem in medical or health care facilities with a high risk for nosocomial infections. Rooms with a continuous airflow, such as operation theaters, are of particular importance due to a possible dissemination and circulation of pathogens including multidrug-resistant microorganisms. In this regard, a cold atmospheric-pressure plasma (CAP) may be a possibility to support usual disinfection procedures due to its decontaminating properties. The aim of this study was to determine the antimicrobial efficacy of a plasma decontamination module that included a dielectric barrier discharge for plasma generation. Experimental parameters such as an airflow velocity of 4.5 m/s and microbial contaminations of approximately 6,000 colony-forming units (cfu)/m3 were used to simulate practical conditions of a ventilation system in an operating theater. The apathogenic microorganism Escherichia coli K12 DSM 11250/NCTC 10538 and the multidrug-resistant strains E. coli 21181 and 21182 (isolated from patients) were tested to determine the antimicrobial efficacy. In summary, the number of cfu was reduced by 31–89% for the tested E. coli strains, whereby E. coli K12 was the most susceptible strain toward inactivation by the designed plasma module. A possible correlation between the number or kind of resistances and susceptibility against plasma was discussed. The inactivation of microorganisms was affected by plasma intensity and size of the plasma treatment area. In addition, the differences of the antimicrobial efficacies caused through the nebulization of microorganisms in front (upstream) or behind (downstream) the plasma source were compared. The presence of ionic wind had no influence on the reduction of the number of cfu for E. coli K12, as the airflow velocity was too high for a successful precipitation, which would be a prerequisite for an increased antimicrobial efficacy. The inactivation of the tested microorganisms confirms the potential of CAP for the improvement of air quality. The scale-up of this model system may provide a novel tool for an effective air cleaning process

Natural phenolic compounds as biofilm inhibitors of multidrug-resistant Escherichia coli – the role of similar biological processes despite structural diversity

Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli

Identifying Multiple Potential Metabolic Cycles in Time-Series from Biolog Experiments

Biolog Phenotype Microarray (PM) is a technology allowing simultaneous screening of the metabolic behaviour of bacteria under a large number of different conditions. Bacteria may often undergo several cycles of metabolic activity during a Biolog experiment. We introduce a novel algorithm to identify these metabolic cycles in PM experimental data, thus increasing the potential of PM technology in microbiology. Our method is based on a statistical decomposition of the time-series measurements into a set of growth models. We show that the method is robust to measurement noise and captures accurately the biologically relevant signals from the data. Our implementation is made freely available as a part of an R package for PM data analysis and can be found at www.helsinki.fi/bsg/software/Biolog_Decomposition.Peer reviewe

Genomic and Phenotypic Analysis of an ESBL-Producing E. coli ST1159 Clonal Lineage From Wild Birds in Mongolia

Background In addition to the broad dissemination of pathogenic extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli in human and veterinary medicine and the community, their occurrence in wildlife and the environment is a growing concern. Wild birds in particular often carry clinically relevant ESBL-producing E. coli. Objectives We analyzed ESBL-producing and non-ESBL-producing E. coli obtained from wild birds in Mongolia to identify phylogenetic and functional characteristics that would explain the predominance of a particular E. coli clonal lineage in this area. Methods We investigated ESBL-producing E. coli using whole-genome sequencing and phylogenetics to describe the population structure, resistance and virulence features and performed phenotypic experiments like biofilm formation and adhesion to epithelial cells. We compared the phenotypic characteristics to non-ESBL-producing E. coli from the same background (Mongolian wild birds) and genomic results to publicly available genomes. Results and Conclusion We found ESBL-producing E. coli sequence type (ST) 1159 among wild birds in Mongolia. This clonal lineage carried virulence features typical for extra-intestinal pathogenic or enterotoxigenic E. coli. Comparative functional experiments suggested no burden of resistance in the ST1159 isolates, which is despite their carriage of ESBL-plasmids. Wild birds will likely disseminate these antibiotic-resistant pathogens further during migration

ESBL-plasmid carriage in E. coli enhances in vitro bacterial competition fitness and serum resistance in some strains of pandemic sequence types without overall fitness cost

Background: Extended spectrum beta lactamase (ESBL)-producing extraintestinal pathogenic Escherichia coli infections are of global interest because of their clinical and economic impact. The ESBL resistance genes disseminate through plasmids, and are found in successful global lineages such as ST131 and ST648. The carriage of plasmids has been suggested to result in a fitness burden, but recently it was shown that ESBL-plasmids enhanced virulence in pandemic ST131 and ST648 lineages without affecting their fitness. Herein, we investigated the influence of ESBL-plasmids on bacterial competition and serum resistance, both of which are essential characteristics of ExPEC during infections. Methods: Triplets of ESBL-plasmid-carrying wildtype (WT), plasmid-cured variant (PCV) and transformant (T) of five ExPEC strains of ST131 and ST648 were used for bacterial competition experiments with colicin-producing commensal E. coli, competitive adhesion experiments and serum survival. In addition, resilience after SDS, acid, osmotic challenges and RNA sequence data were analyzed. Results: In all five strains tested, ESBL-plasmid carriage did not negatively influence E. coli fitness in direct bacterial competition with commensal E. coli in vitro. That is, WTs did not show any disadvantages when compared to their isogenic plasmid-free PCV. For one strain we even found the opposite as PCV17433 was out-competed by a commensal strain, which suggests an even protective role of the ESBL-plasmid carried by the WT17433. Similarly, in the serum-resistance experiments, the PCVs of two strains (PCV17433 and PCV17887) were more sensitive to serum, unlike WTs and Ts. The observed inter-strain differences could be explained by the different genetic content of plasmids carried in those strains. Conclusions: Overall, we found no compelling evidence for an increased burden resulting from the carriage of ESBL-plasmids in the absence of antimicrobial selection pressure in the strains of pandemic ST131 and ST648; rather, the possession of certain ESBL-plasmids was beneficial for some strains in regarding competition fitness and serum survival.Peer Reviewe

Global diversity of enterococci and description of 18 novel species

Bacteria of the genus Enterococcus colonize the guts of diverse animals. Some species have acquired multiple antibiotic resistances on top of a high level of intrinsic resistance and have emerged as leading causes of hospital-associated infection. Although clinical isolates of enterococcal species E. faecalis and E. faecium have been studied with respect to their antibiotic resistances and infection pathogenesis, comparatively little is known about the biology of enterococci in their natural context of the guts of humans and other land animals, including arthropods and other invertebrates. Importantly, little is also known about the global pool of genes already optimized for expression in an enterococcal background with the potential to be readily acquired by hospital adapted strains of E. faecalis and E. faecium , known facile exchangers of mobile genetic elements. We therefore undertook a global study designed to reach into maximally diverse habitats, to establish a first approximation of the genetic diversity of enterococci on Earth. Presumptive enterococci from over 900 diverse specimens were initially screened by PCR using a specific reporter gene that we found to accurately reflect genomic diversity. The genomes of isolates exceeding an operationally set threshold for diversity were then sequenced in their entirety and analyzed. This provided us with data on the global occurrence of many known enterococcal species and their association with various hosts and ecologies and identified 18 novel species expanding the diversity of the genus Enterococcus by over 25%. The 18 novel enterococcal species harbor a diverse array of genes associated with toxins, detoxification, and resource acquisition that highlight the capacity of the enterococci to acquire and adapt novel functions from diverse gut environments. In addition to the discovery and characterization of new species, this expanded diversity permitted a higher resolution analysis of the phylogenetic structure of the Enterococcus genus, including identification of distinguishing features of its 4 deeply rooted clades and genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus , along with new insights into their potential threat to human health

Funktionelle Plasmidanalyse von ESBL-bildenden Escherichia coli der pandemischen Sequenztypen ST131 und ST648

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli occur frequently all over the world, not only in human and veterinary medicine clinical-associated contexts but also in wildlife and the environment, which are considered to be less affected by antimicrobial compounds. Specifically, international high-risk multi-drug resistant clones and lineages of ESBL- associated sequence type (ST) 131 and, to a lesser extent, 648 seem to be of importance. Their possession of ESBL- and non-resistance-factor-carrying plasmids should be more closely examined; the success of certain bacterial clones and lineages might not depend on antimicrobial resistance (AMR) alone. My thesis aimed at investigating the interaction of ESBL-plasmids with chromosomally-encoded, non-resistance features of ST131 and ST648 E. coli. This interaction is important as it might result in benefits for the bacterial host beyond those of AMR such as virulence, fitness and metabolism, yet presents no parallel fitness costs. ESBL-plasmid-carrying wild-type (WT) E. coli strains were compared to a constructed, corresponding ESBL-plasmid “cured” variant (PCV) and to a complementary ESBL-carrying transformant (T) in fitness/metabolic assays, biofilm and motility assays, and RNA sequence analysis. No differences were observed in the strains’ growth or metabolic behaviors. Some differed in biofilm and motility assays, however, exemplified by an enhanced curli and/or cellulose production and a reduced swimming capacity of WTs/Ts compared to the corresponding PCV. RNA sequencing mostly confirmed the phenotypic results on a transcriptomic level, revealing the chromosomally-encoded csgD-pathway to be a key factor. The results clearly indicate that ESBL-plasmid carriage does not necessarily lead to a fitness/metabolic disadvantage for the bacterial host. On the contrary, the results suggest that an interaction of ESBL-plasmids with the bacterial host’s chromosome in some strains, especially in terms of non-resistance-associated features, presumably contributes to the pandemic success of some isolates of ESBL-producing E. coli clones and lineages in various hosts and habitats, also beyond high antimicrobial selection pressures.Extended-spektrum beta-Laktamase (ESBL)-bildende Escherichia (E.) coli kommen häufig weltweit nicht nur in human- und veterinärmedizinisch klinisch- assoziierten Kontexten, sondern auch in Wildtieren und der Umwelt vor. Bei letzteren wird angenommen, dass diese durch antimikrobielle Wirkstoffe weit weniger beeinflusst sind. Besonders internationale Hoch-Risiko Klone und Linien des ESBL-assoziierten Sequenztyps (ST) 131 und in geringerem Maß 648 scheinen von Bedeutung zu sein. Ihre Trägerschaft von ESBL- und nicht- Resistenzfaktoren-tragenden Plasmiden sollte genauer untersucht werden, da der Erfolg von bestimmten bakteriellen Klonen und Linien nicht allein von einer antimikrobiellen Resistenz abhängig zu sein scheint. Meine Dissertation hatte insbesondere zum Ziel, die Interaktion von ESBL-Plasmiden mit chromosomal- kodierten Nichtresistenzfaktoren von ST131 und ST648 E. coli zu untersuchen. Bei dieser Interaktion außerhalb antimikrobieller Resistenzen handelt es sich zum Beispiel um Virulenz, Fitness und Metabolismus, welche in Vorteilen für den bakteriellen Wirt resultieren und keine parallelen Fitnesskosten verursachen. Die ESBL-Plasmid-tragenden Wild-typ (WT) E. coli Stämme wurden mit einer konstruierten, korrespondierenden ESBL-Plasmid „gecurten“ Variante (PCV) und einer komplementären ESBL-tragenden Transformante (T) in Fitness/Metabolismus Assays, Biofilm- und Motilitätstests sowie in einer RNA Sequenzanalyse verglichen. Es wurden keine Unterschiede der Stämme bezüglich ihres Wachstums- oder metabolischen Verhaltens festgestellt, allerdings unterschieden sich einige in den Biofilm- und Motilitätstests, exemplifiziert durch eine erhöhte Curli und/oder Zellulose Bildung und einer reduzierten Schwimmkapazität der WTs/Ts verglichen zur korrespondierenden PCV. Die RNA Sequenzanalyse bestätigte zu großen Teilen die phänotypischen Ergebnisse auf Transkriptomebene und zeigte den chromosomal-kodierten csgD-Pfad als Schlüsselfaktor auf. Die Resultate indizieren klar, dass eine ESBL-Plasmid Trägerschaft nicht notwendigerweise zu einem Fitness/Metabolismus Nachteil für den bakteriellen Wirt führt. Im Gegenteil, bei manchen Stämmen trägt eine Interaktion von ESBL-Plasmiden mit dem Chromosom des bakteriellen Wirts, speziell bezüglich Nichtresistenz-assoziierter Faktoren, vermutlich zum pandemischen Erfolg einiger Isolate von ESBL-produzierender E. coli Klonen und Linien in unterschiedlichen Wirten und Habitaten bei, auch außerhalb eines antimikrobiellen Selektionsdrucks