Search

Abstract The global spread of multidrug-resistant Escherichia coli threatens human, animal, and environmental health. Despite the recognition of water bodies as potential reservoirs, research on antimicrobial resistance (AMR) and antibiotic residues in the Baltic Sea is limited. In this study, we examined the presence of ESBL-producing E. coli in surface water in northeastern Germany. We then conducted phenotypic and genotypic analyses of the strains, which encompassed AMR and heavy metal/metalloid tolerance, biofilm formation, and detailed plasmid examination. In addition, we used solid-phase extraction with ultra-high performance liquid chromatography and mass spectrometry to quantify antibiotic residues. Our analysis identified 30 ESBL-producing E. coli isolates, some of which were associated with clinically relevant high-risk clonal lineages. We detected antibiotic residues in the environmental samples, but the antibiotic concentrations were significantly lower compared to wastewater samples. These results suggest that the Baltic Sea surface water could serve as a reservoir for ESBL-producing E. coli and clinically relevant antibiotics.

BackgroundConvergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307.MethodsHypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics.ResultsOur results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways.ConclusionThis study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

IntroductionHorse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum β-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited.MethodsWe collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1–V2 gene sequencing.ResultsWe included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1.ConclusionColic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses.

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.

Abstract Background Klebsiella pneumoniae, which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage. Methods We computationally analyzed a large collection of K. pneumoniae ST395 genomes (n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features. Results Clustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved different K. pneumoniae donors and affected, inter alia, capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of “hybrid” resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype. Conclusions To the best of our knowledge, this is the first study that investigated a large international collection of K. pneumoniae ST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.

The first steps of the global process of photosynthesis take place in specialized membrane pigment–protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium Rhodobacter sphaeroides, a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment–protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC. It has previously been shown that the replacement of Phe M197 by His strongly affects the oxidation potential of P (Em P/P+), increasing its value by 125 mV, as well as increasing the thermal stability of RC and its stability in response to external pressure. The crystal structures of F(M197)H RC at high resolution obtained using various techniques presented in this report clarify the optical and electrochemical properties of the primary electron donor and the increased resistance of the mutant complex to denaturation. The electron-density maps are consistent with the donation of a hydrogen bond from the imidazole group of His M197 to the C2-acetyl carbonyl group of BChl PB. The formation of this hydrogen bond leads to a considerable out-of-plane rotation of the acetyl carbonyl group and results in a 1.2 Å shift of the O atom of this group relative to the wild-type structure. Besides, the distance between BChl PA and PB in the area of pyrrole ring I was found to be increased by up to 0.17 Å. These structural changes are discussed in association with the spectral properties of BChl dimer P. The electron-density maps strongly suggest that the imidazole group of His M197 accepts another hydrogen bond from the nearest water molecule, which in turn appears to form two more hydrogen bonds to Asn M195 and Asp L155. As a result of the F(M197)H mutation, BChl PB finds itself connected to the extensive hydrogen-bonding network that pre-existed in wild-type RC. Dissimilarities in the two hydrogen-bonding networks near the M197 and L168 sites may account for the different changes of the Em P/P+ in F(M197)H and H(L168)F RCs. The involvement of His M197 in the hydrogen-bonding network also appears to be related to stabilization of the F(M197)H RC structure. Analysis of the experimental data presented here and of the data available in the literature points to the fact that the hydrogen-bonding networks in the vicinity of BChl dimer P may play an important role in fine-tuning the redox properties of the primary electron donor.

This communication introduces the first-time application of high-resolution continuum-source molecular absorption spectrometry (HR CS MAS) for the quantification of a peptide. The graphite furnace technique was employed and the tripeptide glutathione (GSH) served as a model compound. Based on measuring sulfur in terms of carbon monosulfide (CS), a method was elaborated to analyze aqueous solutions of GSH. The most prominent wavelength of the CS molecule occurred at 258.0560 nm and was adduced for monitoring. The methodological development covered the optimization of the pyrolysis and vaporization temperatures. These were found optimally to be 250 °C and 2250 °C, respectively. Moreover, the effect of modifiers (zirconium, calcium, magnesium, palladium) on the absorption signals was investigated. The best results were obtained after permanent coating of the graphite tube with zirconium (total amount of 400 μg) and adding a combination of palladium (10 µL, 10 g L−1) and calcium (2 µL, 1 g L−1) as a chemical modifier to the probes (10 µL). Aqueous standard samples of GSH were used for the calibration. It showed a linear range of 2.5–100 µg mL−1 sulfur contained in GSH with a correlation coefficient R2 > 0.997. The developed method exhibited a limit of detection (LOD) and quantification (LOQ) of 2.1 µg mL−1 and 4.3 µg mL−1 sulfur, respectively. The characteristic mass accounted for 5.9 ng sulfur. The method confirmed the general suitability of MAS for the analysis of an oligopeptide. Thus, this study serves as groundwork for further development in order to extend the application of classical atomic absorption spectrometry (AAS).

ABSTRACT The ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages. IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

The goal of this study was to assess the anticancer efficacy of chlorojanerin against various cancer cells. The effects of chlorojanerin on cell cytotoxicity, cell cycle arrest, and cell apoptosis were examined using MTT assay, propidium iodide staining, and FITC Annexin V assay. RT-PCR was employed to determine the expression levels of apoptosis-related genes. Furthermore, docking simulations were utilized to further elucidate the binding preferences of chlorojanerin with Bcl-2. According to MTT assay, chlorojanerin inhibited the proliferation of all tested cells in a dose-dependent manner with a promising effect against A549 lung cancer cells with an IC50 of 10 µM. Cell growth inhibition by chlorojanerin was linked with G2/M phase cell cycle arrest in A549 treated cells. Flow cytometry analysis indicated that the proliferation inhibition effect of chlorojanerin was associated with apoptosis induction in A549 cells. Remarkably, chlorojanerin altered the expression of many genes involved in apoptosis initiation. Moreover, we determined that chlorojanerin fit into the active site of Bcl-2 according to the molecular docking study. Collectively, our results demonstrate that chlorojanerin mediated an anticancer effect involving cell cycle arrest and apoptotic cell death and, therefore, could potentially serve as a therapeutic agent in lung cancer treatment.

Abstract Background Antibiotic-resistant Klebsiella pneumoniae are a major cause of hospital- and community-acquired infections, including sepsis, liver abscess, and pneumonia, driven mainly by the emergence of successful high-risk clonal lineages. The K. pneumoniae sequence type (ST) 307 lineage has appeared in several different parts of the world after first being described in Europe in 2008. From June to October 2019, we recorded an outbreak of an extensively drug-resistant ST307 lineage in four medical facilities in north-eastern Germany. Methods Here, we investigated these isolates and those from subsequent cases in the same facilities. We performed whole-genome sequencing to study phylogenetics, microevolution, and plasmid transmission, as well as phenotypic experiments including growth curves, hypermucoviscosity, siderophore secretion, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance for an in-depth characterization of this outbreak clone. Results Phylogenetics suggest a homogenous phylogram with several sub-clades containing either isolates from only one patient or isolates originating from different patients, suggesting inter-patient transmission. We identified three large resistance plasmids, carrying either NDM-1, CTX-M-15, or OXA-48, which K. pneumoniae ST307 likely donated to other K. pneumoniae isolates of different STs and even other bacterial species (e.g., Enterobacter cloacae) within the clinical settings. Several chromosomally and plasmid-encoded, hypervirulence-associated virulence factors (e.g., yersiniabactin, metabolite transporter, aerobactin, and heavy metal resistance genes) were identified in addition. While growth, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance were comparable to several control strains, results from siderophore secretion and hypermucoviscosity experiments revealed superiority of the ST307 clone, similar to an archetypical, hypervirulent K. pneumoniae strain (hvKP1). Conclusions The combination of extensive drug resistance and virulence, partly conferred through a “mosaic” plasmid carrying both antibiotic resistance and hypervirulence-associated features, demonstrates serious public health implications.

Extended spectrum beta-lactamase (ESBL)-producing Escherichia coli are an emerging problem in veterinary and human medicine. Our study concentrated on the estimation of the prevalence and factors associated with the carriage of ESBL-producing E. coli in dogs who visited a veterinary clinic in northern Germany in 2017. For this reason, 1000 patients (healthy and sick dogs) were tested, resulting in 1000 samples originating from rectal swabs. Additional data were collected using a self-reported questionnaire that was completed by the dog owner. Factors associated with ESBL carriage were considered for further modeling if p < 0.05 using a two-sided Fisher test. Using a backward elimination procedure, the variables for the final multivariable logistic regression model were identified. In total, 8.9% of the dogs tested were positive for carriage of ESBL-producing E. coli. Seven factors were associated with the colonization of dogs with ESBL-E. coli within the multivariable model, namely husbandry system (p = 0.0019, OR = 3.00; 95% CI: 1.50–6.00), contact with puppies (p = 0.0044, OR = 2.43; 95% CI: 1.32–4.46), feeding of raw meat (p = 0.011, OR = 2.28; 95% CI: 1.21–4.31), food residues (p = 0.0151, OR = 2.31; 95% CI: 1.18–4.53) and food supplements (p = 0.0487, OR = 0.426; 95% CI: 0.18–0.96), and antibiotic treatments of dogs (p = 0.0005, OR = 3.030; 95% CI: 1.62–5.68) or owners (p = 0.041, OR = 2.74; 95% CI: 1.04–7.19) prior to the study. These factors refer to the animals themselves as well as to the owners and their habits or medical treatments. Although the causality and direction of transmission from owners to their dogs cannot be proven, the factor of antibiotic treatment of the owner is clearly associated with the dog’s status.

Multidrug-resistant (MDR) Enterobacterales, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, not only emerge in healthcare settings but also in other habitats, such as livestock and wildlife. The spread of these pathogens, which often combine resistance with high-level virulence, is a growing problem, as infections have become increasingly difficult to treat. Here, we investigated the occurrence of ESBL-producing E. coli and K. pneumoniae in fecal samples from two black-headed gull colonies breeding on two nature conservation islands in Western Pomerania, Germany. In addition to cloacal samples from adult birds (n = 211) and their nestlings (n = 99) during the 2021 breeding season, collective fecal samples (n = 29) were obtained. All samples were screened for ESBL producers, which were then subjected to whole-genome sequencing. We found a total of 12 ESBL-producing E. coli and K. pneumoniae consisting of 11 E. coli and 1 K. pneumoniae, and including the international high-risk E. coli sequence types (ST)131, ST38, and ST58. Eight of the investigated strains had a MDR genotype and carried a large repertoire of virulence-associated genes, including the pap operon, which is important for urinary tract infections. In addition, we identified many genes associated with adherence, biofilm formation, iron uptake, and toxin production. Finally, our analysis revealed the close phylogenetic relationship of ST38 strains with genomes originating from human sources, underlining their zoonotic and pathogenic character. This study highlights the importance of the One Health approach, and thus the interdependence between human and animal health and their surrounding environment.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, `naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

The colonization of broilers with extended-spectrum β-lactamase- (ESBL-) and plasmid-mediated AmpC β-lactamase- (pAmpC-) producing Enterobacteriaceae has been extensively studied. However, only limited data on intervention strategies to reduce the colonization throughout the fattening period are available. To investigate practically relevant management measures for their potential to reduce colonization, a recently published seeder-bird colonization model was used. Groups of 90 broilers (breed Ross 308) were housed in pens under conventional conditions (stocking of 39 kg/m2, no enrichment, water and feed ad libitum). Tested measures were investigated in separate trials and included (I) an increased amount of litter in the pen, (II) the reduction of stocking density to 25 kg/m2, and (III) the use of an alternative broiler breed (Rowan x Ranger). One-fifth of ESBL- and pAmpC- negative broilers (n = 18) per group were orally co-inoculated with two E. coli strains on the third day of the trial (seeder). One CTX-M-15-positive E. coli strain (ST410) and one CMY-2 and mcr-1-positive E. coli strain (ST10) were simultaneously administered in a dosage of 102 cfu. Colonization of all seeders and 28 non-inoculated broilers (sentinel) was assessed via cloacal swabs during the trials and a final necropsy at a target weight of two kilograms (= d 36 (control, I-II), d 47 (III)). None of the applied intervention measures reduced the colonization of the broilers with both the ESBL- and the pAmpC- producing E. coli strains. A strain-dependent reduction of colonization for the ESBL- producing E. coli strain of ST410 by 2 log units was apparent by the reduction of stocking density to 25 kg/m2. Consequently, the tested management measures had a negligible effect on the ESBL- and pAmpC- colonization of broilers. Therefore, intervention strategies should focus on the prevention of ESBL- and pAmpC- colonization, rather than an attempt to reduce an already existing colonization.

In the search for alternative treatment options for infections with multi-resistant germs, traditionally used medicinal plants are currently being examined more intensively. In this study, the antimicrobial and anti-biofilm activities of 14 herbal drugs were investigated. Nine of the tested drugs were traditionally used in Europe for treatment of local infections. For comparison, another five drugs monographed in the European Pharmacopoeia were used. Additionally, the total tannin and flavonoid contents of all tested drugs were analyzed. HPLC fingerprints were recorded to obtain further insights into the components of the extracts. The aim of the study was to identify herbal drugs that might be useable for treatment of infectious diseases, even with multidrug resistant E. coli, and to correlate the antimicrobial activity with the total content of tannins and flavonoids. The agar diffusion test and anti-biofilm assay were used to evaluate the antimicrobial potential of different extracts from the plants. Colorimetric methods (from European Pharmacopeia) were used for determination of total tannins and flavonoids. The direct antimicrobial activity of most of the tested extracts was low to moderate. The anti-biofilm activity was found to be down to 10 µg mL−1 for some extracts. Tannin contents between 2.2% and 10.4% of dry weight and total flavonoid contents between 0.1% and 1.6% were found. Correlation analysis indicates that the antimicrobial and the anti-biofilm activity is significantly (p < 0.05) dependent on tannin content, but not on flavonoid content. The data analysis revealed that tannin-rich herbal drugs inhibit pathogens in different ways. Thus, some of the tested herbal drugs might be useable for local infections with multi-resistant biofilm-forming pathogens. For some of the tested drugs, this is the first report about anti-biofilm activity, as well as total tannin and flavonoid content.

BackgroundIn 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.ObjectivesWe 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.MethodsWe 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 ConclusionWe 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.

Abstract 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.

Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC50 values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC50 values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.

Extended-spectrum beta-lactamase- (ESBL-) and AmpC beta-lactamase- (AmpC-) producing Enterobacteriaceae pose a risk for both human and animal health. For livestock, highest prevalences have been reported in broiler chickens, which are therefore considered as a reservoir of multidrug-resistant bacteria. The possibility of transfer to humans either by a close contact to colonized broiler flocks or through contaminated retail meat results in the necessity to develop intervention measures for the entire broiler production chain. In this regard, a basic understanding of the colonization process is mandatory including the determination of the minimal bacterial load leading to a persistent colonization of broiler chickens. Therefore, we conducted a bivalent broiler colonization study close to real farming conditions without applying any antimicrobial selection pressure. ESBL- and AmpC- negative broiler chickens (Ross 308) were co- colonized on their third day of life with two strains: one CTX-M-15-producing Escherichia coli-ST410 and one CMY-2/mcr-1-positive E. coli-ST10. Colonization was assessed by cloacal swabs over the period of the trial, starting 24 h post inoculation. During the final necropsy, the contents of crop, jejunum, cecum, and colon were quantified for the occurrence of both bacterial strains. To define the minimal oral colonization dosage 104 to 101 colony forming units (cfu) were orally inoculated to four separately housed broiler groups (each n = 19, all animals inoculated) and a dosage of already 101 cfu E. coli led to a persistent colonization of all animals of the group after 3 days. To assure stable colonization, however, a dosage of 102 cfu E. coli was chosen for the subsequent seeder-bird trial. In the seeder-bird trial one fifth of the animals (seeder, n = 4) were orally inoculated and kept together with the non-inoculated animals (sentinel, n = 16) to mimic the route of natural infection. After 35 days of trial, all animals were colonized with both E. coli strains. Given the low colonization dosage and the low seeder/sentinel ratio, the rapid spread of ESBL- and AmpC- producing Enterobacteriaceae in conventional broiler farms currently seems inevitably resulting in an urgent need for the development of intervention strategies to reduce colonization of broilers during production.

Introduction Diabetes mellitus (DM) has had a wide-ranging impact on healthcare politics. Secondary diseases and complications caused by diabetes are relevant cost and utilisation factors in the healthcare system. For decades, diabetes self-management education (DSME) has played a major role in the treatment of patients with type 2 DM (T2DM). The aim of this training is to empower patients to actively influence their diabetes process by gaining knowledge about health-related behaviours, such as healthy nutrition and exercise, and cardiovascular risks. The aim of the project is to analyse the practice of structured diabetes education and the effects of different learning types of participants. This project focuses on the needs of socioeconomically deprived patients and aims to improve DSME for this group. This patient group has a higher prevalence of T2DM, more complications and worse therapy-relevant parameters.Methods and analysis The study will be conducted as a prospective longitudinal study. Patients will be recruited in outpatient physician offices over a period of 12 months. Patients will be included if they are 18 years and older, have T2DM and are scheduled to participate in DSME for the first time. A pseudonymised, written survey with standardised questionnaires will be administered. The data will be analysed using inferential statistical methods, such as correlation analysis, regression models and variance analytical designs.Ethics and dissemination The study will be carried out following the principles of the Declaration of Helsinki and good scientific standards. Ethical approval of the Ethics Review Committee of the Medical Faculty at Martin-Luther-University, Halle-Wittenberg, was obtained. All participants in the study will receive comprehensive information and will be included after written informed consent is obtained. The results will be published in international peer-reviewed journals and presented at several congresses.Trial registration number DRKS00016630

ABSTRACT In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel electrophoresis (PFGE) hybridization experiments. WGS data of these isolates were compared with those for the previously isolated VIM-1-producing Salmonella Infantis isolates from pigs and poultry. Among 103 samples, one Salmonella Goldcoast isolate, one Salmonella Infantis isolate, and one Enterobacter cloacae isolate carrying the blaVIM-1 gene were detected. Comparative WGS analysis revealed that the blaVIM-1 gene was part of a particular Tn21-like transposable element in all isolates. It was located on IncHI2 (ST1) plasmids of ∼290 to 300 kb with a backbone highly similar (98 to 100%) to that of reference pSE15-SA01028. SNP analysis revealed a close relationship of all VIM-1-positive S. Infantis isolates described since 2011. The findings of this study demonstrate that the occurrence of the blaVIM-1 gene in German livestock is restricted neither to a certain bacterial species nor to a certain Salmonella serovar but is linked to a particular Tn21-like transposable element located on transferable pSE15-SA01028-like IncHI2 (ST1) plasmids, being present in all of the investigated isolates from 2011 to 2017. IMPORTANCE Carbapenems are considered one of few remaining treatment options against multidrug-resistant Gram-negative pathogens in human clinical settings. The occurrence of carbapenemase-producing Enterobacteriaceae in livestock and food is a major public health concern. Particularly the occurrence of VIM-1-producing Salmonella Infantis in livestock farms is worrisome, as this zoonotic pathogen is one of the main causes for human salmonellosis in Europe. Investigations on the epidemiology of those carbapenemase-producing isolates and associated mobile genetic elements through an in-depth molecular characterization are indispensable to understand the transmission of carbapenemase-producing Enterobacteriaceae along the food chain and between different populations to develop strategies to prevent their further spread.

Minimal inhibitory concentration-based pharmacokinetic/pharmacodynamic (PK/PD) indices are commonly applied to antibiotic dosing optimisation, but their informative value is limited, as they do not account for bacterial growth dynamics over time. We aimed to comprehensively characterise the exposure–effect relationship of levofloxacin against Escherichia coli and quantify strain-specific characteristics applying novel PK/PD parameters. In vitro infection model experiments were leveraged to explore the exposure–effect relationship of three clinical Escherichia coli isolates, harbouring different genomic fluoroquinolone resistance mechanisms, under constant levofloxacin concentrations or human concentration–time profiles (≤76 h). As an exposure metric, the ‘cumulative area under the levofloxacin–concentration time curve’ was determined. The antibiotic effect was assessed as the ‘cumulative area between the growth control and the bacterial-killing and -regrowth curve’. PK/PD modelling was applied to characterise the exposure–effect relationship and derive novel PK/PD parameters. A sigmoidal Emax model with an inhibition term best characterised the exposure–effect relationship and allowed for discrimination between two isolates sharing the same MIC value. Strain- and exposure-pattern-dependent differences were captured by the PK/PD parameters and elucidated the contribution of phenotypic adaptation to bacterial regrowth. The novel exposure and effect metrics and derived PK/PD parameters allowed for comprehensive characterisation of the isolates and could be applied to overcome the limitations of the MIC in clinical antibiotic dosing decisions, drug research and preclinical development.

To increase our understanding of bacterial intestinal colonization in animal populations lacking substantial anthropogenic influence we studied the diversity of E. coli in cormorants from the pristine West-Mongolian steppe. E. coli were isolated from individual birds of two cormorant colonies located on small islands in lakes at least 100 km away from human settlements. Diversity of the isolates was studied using pulsed-field gel electrophoresis (PFGE). 137 isolates of cormorant colony-1 and 75 isolates of cormorant colony-2 resulted in 60 and 33 PFGE types, respectively. Representative strains of each PFGE type were analyzed via PCR in terms of phylogroups and extraintestinal virulence-associated genes (exVAGs). Bacterial adhesion to the chicken intestinal cell line CHIC-8E11 and antimicrobial resistance was also determined. Most isolates belonged to phylogroup B1 (68.3%) followed by B2 and E with B2 harboring the highest total number of exVAGs per isolate. Unexpectedly, a PFGE type with relatively few exVAGs displayed the highest isolation frequency, also showing a high adhesion rate. Comparative analysis of exVAGs to other E. coli populations of wildlife origin revealed that the secreted autotransporter toxin encoding sat gene was only present in cormorants. Overall, E. coli in cormorants maintained a high diversity under minimal anthropogenic influences, which likely enables intestinal colonization.

ESBL-producing Klebsiella pneumoniae (K. pneumoniae) represent an increasing problem both in human and veterinary medicine. As SHV-2 - encoding K. pneumoniae were recently detected in the broiler production we were interested in investigating a possible transmission along the broiler production chain and furthermore, in evaluating their possible impact on human health. Therefore, 41 ESBL-producing K. pneumoniae originating from a parent flock, from the hatcherys' environment during the hatching of that parent flocks' chickens, and from an associated fattening flock were investigated on an Illumina Miseq. Whole genome sequences were analyzed concerning their MLST-type, cgMLST-type, genotypic and phenotypic resistance, plasmid profiles and virulence genes. Irrespective of the origin of isolation all investigated isolates were multi-drug resistant, harbored the same ESBL-gene blaSHV−2, shared the same sequence type (ST3128) and displayed 100% similarity in core genome multilocus sequence typing (cgMLST). In addition, in silico plasmid typing found several Inc/Rep types associated with ESBL-plasmids. Summarizing, identical clones of SHV-2—producing K. pneumoniae were detected in different stages of the industrial broiler production in one out of seven investigated broiler chains. This proves the possibility of pseudo-vertical transmission of multi-resistant human pathogens from parent flocks to hatcheries and fattening flocks. Furthermore, the importance of cross-contamination along the production chain was shown. Although the ESBL-producing K. pneumoniae clone detected here in the broiler production has not been associated with clinical settings so far, our findings present a potential public health threat.

Ascariasis is a widespread soil-transmitted helminth infection caused by the intestinal roundworm Ascaris lumbricoides in humans, and the closely related Ascaris suum in pigs. Progress has been made in understanding interactions between helminths and host immune cells, but less is known concerning the interactions of parasitic nematodes and the host microbiota. As the host microbiota represents the direct environment for intestinal helminths and thus a considerable challenge, we studied nematode products, including excretory-secretory products (ESP) and body fluid (BF), of A. suum to determine their antimicrobial activities. Antimicrobial activities against gram-positive and gram-negative bacterial strains were assessed by the radial diffusion assay, while effects on biofilm formation were assessed using the crystal violet static biofilm and macrocolony assays. In addition, bacterial neutralizing activity was studied by an agglutination assay. ESP from different A. suum life stages (in vitro-hatched L3, lung-stage L3, L4, and adult) as well as BF from adult males were analyzed by mass spectrometry. Several proteins and peptides with known and predicted roles in nematode immune defense were detected in ESP and BF samples, including members of A. suum antibacterial factors (ASABF) and cecropin antimicrobial peptide families, glycosyl hydrolase enzymes such as lysozyme, as well as c-type lectin domain-containing proteins. Native, unconcentrated nematode products from intestine-dwelling L4-stage larvae and adults displayed broad-spectrum antibacterial activity. Additionally, adult A. suum ESP interfered with biofilm formation by Escherichia coli, and caused bacterial agglutination. These results indicate that A. suum uses a variety of factors with broad-spectrum antibacterial activity to affirm itself within its microbe-rich environment in the gut.

Carbapenems are last-resort antibiotics used in human medicine. The increased detection of carbapenem-resistant Enterobacteriaceae (CRE) is therefore worrying. In 2011 we reported the first livestock-associated VIM-1-producing Salmonella (S.) enterica serovar Infantis (R3) isolate from dust, sampled in a German chicken fattening farm. Due to this observation we retrospectively investigated more than 536 stored bacterial cultures, isolated from 45 chicken fattening farms during the years 2011 and 2012. After a non-selective overnight incubation, the bacteria were transferred to selective media. Escherichia (E.) coli and Salmonella growing on these media were further investigated, including antibiotic susceptibility testing, carbapenemase gene screening and whole genome sequencing (WGS). In total, four CRE were found in three out of 45 investigated farms: Besides R3, one additional Salmonella (G-336-1a) as well as two E. coli isolates (G-336-2, G-268-2). All but G-268-2 harbored the blaVIM-1 gene. Salmonella isolates R3 and G-336-1 were closely related although derived from two different farms. All three blaVIM-1-encoding isolates possessed identical plasmids and the blaVIM-1- containing transposon showed mobility at least in vitro. In isolate G-268-2, the AmpC beta-lactamase gene blaCMY-2 but no known carbapenemase gene was identified. However, a transfer of the phenotypic resistance was possible. Furthermore, G-268-2 contained the mcr-1 gene, combining phenotypical carbapenem- as well as colistin resistance in one isolate. Carbapenem-resistant Enterobacteriaceae have been found in three out of 45 investigated chicken flocks. This finding is alarming and emphasizes the importance of intervention strategies to contain the environmental spread of resistant bacteria in animals and humans.

High-risk ESBL-producing Enterobacteriaceae (ESBL-E) have been described in wild birds and rodents worldwide. Rats are of special interest not only due to their indicator role for environmental pollution with multi-resistant bacteria but also as possible infection source. Data on the presence of high-risk ESBL-E in urban wildlife from Africa remain scarce, however. Twenty-nine animals from three different rat (Rattus) species were captured in the city of Conakry (Guinea, West Africa) in 2015. Rectal swabs were analyzed for ESBL-E using selective media. Species typing and phenotypic antimicrobial resistance analysis to broad-spectrum beta-lactams and other classes of antimicrobials was performed for Enterobacteriaceae-like isolates using the VITEK®2 system (BioMérieux, Germany). Confirmed ESBL-producing E. coli and K. pneumoniae were whole-genome sequenced and resistance genes, phylogenetic background and genes related to bacterial fitness and virulence were analyzed. In total, six of twenty-nine rats (20%) carried ESBL-E (K. pneumoniae and E. coli). All ESBL-producers were multi-drug resistant with blaCTX−M−15 as the dominating ESBL-type. Interestingly, ESBL-associated clonal lineages E. coli ST38 and K. pneumoniae ST307 were found. The ESBL-plasmid in K. pneumoniae ST307 revealed high sequence similarities to pKPN3-307_TypeC, a >200 kbp IncFII plasmid originating from a human clinical ST307 isolate. This was in contrast to the core genome: the rat isolate was distantly related to the human clinical ST307 isolate (27 SNPs/Mbp). In addition, we identified π-fimbrial, capsule 2, and glycogen synthesis clusters in the rodent ST307 isolate, whose involvement in the adaptation to survival outside the host and in human urinary tracts has been suggested. Our results demonstrate the presence of clinically relevant, ESBL-producing K. pneumoniae ST307 and E. coli ST38 clonal lineages in an urban West African rat population. The human community is likely the initial source of ESBL-E however, rats might function as infection source and transmission hub, accelerated by frequent interactions at a human-wildlife interface.

High levels of zinc oxide are used frequently as feed additive in pigs to improve gut health and growth performance and are still suggested as an alternative to antimicrobial growth promoters. However, we have recently described an increase of multi-resistant E. coli in association to zinc feeding in piglets. This previous study focused on clonal diversity of E. coli, observing the effect on multi-resistant strains by chance. To shed further light into this highly important topic and falsify our previous findings, we performed a zinc pig feeding trial where we specifically focused on in-depth analysis of antimicrobial resistant E. coli. Under controlled experimental conditions, piglets were randomly allocated to a high dietary zinc (zinc group) and a background zinc feeding group (control group). At different ages samples were taken from feces, digesta, and mucosa and absolute E. coli numbers were determined. A total of 2665 E. coli isolates were than phenotypically tested for antimicrobial resistance and results were confirmed by minimum inhibitory concentration testing for random samples. In piglets fed with high dietary zinc, we detected a substantial increase of multi-resistant E. coli in all gut habitats tested, ranging from 28.9-30.2% multi-resistant E. coli compared to 5.8-14.0% in the control group. This increase was independent of the total number of E. coli. Interestingly, the total amount of the E. coli population decreased over time. Thus, the increase of the multi-resistant E. coli populations seems to be linked with persistence of the resistant population, caused by the influence of high dietary zinc feeding. In conclusion, these findings corroborate our previous report linking high dietary zinc feeding of piglets with the occurrence of antimicrobial resistant E. coli and therefore question the feeding of high dietary zinc oxide as alternative to antimicrobial growth promoters.

Pathogens frequently associated with multi-drug resistant (MDR) phenotypes, including extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and Acinetobacter baumannii isolated from horses admitted to horse clinics, pose a risk for animal patients and personnel in horse clinics. To estimate current rates of colonization, a total of 341 equine patients were screened for carriage of zoonotic indicator pathogens at hospital admission. Horses showing clinical signs associated with colic (n = 233) or open wounds (n = 108) were selected for microbiological examination of nostril swabs, faecal samples and wound swabs taken from the open wound group. The results showed alarming carriage rates of Gram-negative MDR pathogens in equine patients: 10.7% (34 of 318) of validated faecal specimens were positive for ESBL-E (94%: ESBL-producing Escherichia coli), with recorded rates of 10.5% for the colic and 11% for the open wound group. 92.7% of the ESBL-producing E. coli were phenotypically resistant to three or more classes of antimicrobials. A. baumannii was rarely detected (0.9%), and all faecal samples investigated were negative for Salmonella, both directly and after two enrichment steps. Screening results for the equine nostril swabs showed detection rates for ESBL-E of 3.4% among colic patients and 0.9% in the open wound group, with an average rate of 2.6% (9/340) for both indications. For all 41 ESBL-producing E. coli isolated, a broad heterogeneity was revealed using pulsed-field gel electrophoresis (PFGE) patterns and whole genome sequencing (WGS) -analysis. However, a predominance of sequence type complex (STC)10 and STC1250 was observed, including several novel STs. The most common genes associated with ESBL-production were identified as blaCTX-M-1 (31/41; 75.6%) and blaSHV-12 (24.4%). The results of this study reveal a disturbingly large fraction of multi-drug resistant and ESBL-producing E. coli among equine patients, posing a clear threat to established hygiene management systems and work-place safety of veterinary staff in horse clinics.

The increased presence of clinically relevant multidrug resistant bacteria in natural environments is an emerging challenge for global health care. Little is known regarding the occurrence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) from environmental sentinels in Pakistan. The goal of the current study was to gain insights into the prevalence and phylogenetic relationships of ESBL-E. coli recovered from wild birds in Pakistan during winter migration. After initial screening of fecal samples on selective chromogenic agar, ESBL-E.coli were analyzed phenotypically using the Vitek-2 automated system. Genotypic characterization was performed using whole genome sequencing (WGS) followed by an in-depth in silico analysis. Of 150 birds screened, 26 (17.3%) were fecal carriers of ESBL-E. coli. Of these, 88.4% isolates exhibited multidrug resistance (MDR) phenotypes. Resistance to cefotaxime, ceftazidime, ampicillin, doxycycline, tetracycline and sulfamethoxazole/trimethoprim (CTX-CAZ-AM-DC-TE-SXT) represented the most common pattern of MDR (76.9%). WGS data analysis found blaCTX-M-15 as the predominant ESBL genotype (92.3%). Other genes encoding resistance to sulfonamides (sul1/sul2/sul3), aminoglycosides (strA, strB, aadA1, aadA2, aadA5, aac(3)-IId-like, aac(3)-IVa-like and aph(4)-Ia), trimethoprim (dfrA14 or dfrA17), tetracyclines [tet(A)/tet(B)], and fluoroquinolones (qnrS1) were detected commonly, often encoded on IncF-type plasmids (76.9%). ESBL-E. coli were assigned to 17 different sequence types (STs) of which ST10 and ST7097 (4 isolates each) were the most abundant followed by ST4720, ST93, and ST1139 (2 isolates each). Core-genome phylogeny of the isolates found low numbers (0–29) of single nucleotide polymorphisms (SNPs) in isolates belonged to ST7097 originated from two different locations (Chashma barrage and Rasul barrage). Similar trends were found among isolates belong to ST1139. In addition, WGS-based plasmid typing and S1-digestion found plasmids of the same pMLST type (IncF[F-:A-:B53]) and similar sizes in different bacterial and avian hosts suggesting horizontal gene transfer as another possibility for the spread of ESBL-E. coli in avian wildlife in Pakistan.

Bats are suspected to be a reservoir of several bacterial and viral pathogens relevant to animal and human health, but studies on Escherichia coli in these animals are sparse. We investigated the presence of E. coli in tissue samples (liver, lung and intestines) collected from 50 fruit bats of five different species (Eidolon helvum, Epomops franqueti, Hypsignathus monstrosus, Myonycteris torquata, Rousettus aegyptiacus) of two different areas in the Republic of Congo between 2009 and 2010. To assess E. coli pathotypes and phylogenetic relationships, we determined the presence of 59 virulence associated genes and multilocus sequence types (STs). Isolates were further tested for their susceptibility to several antimicrobial substances by agar disk diffusion test and for the presence of an Extended-Spectrum Beta-Lactamase phenotype. E. coli was detected in 60% of the bats analysed. The diversity of E. coli strains was very high, with 37 different STs within 40 isolates. Occasionally, we detected sequence types (e.g. ST69, ST127, and ST131) and pathotypes (e.g. ExPEC, EPEC and atypical EPEC), which are known pathogens in human and/or animal infections. Although the majority of strains were assigned to phylogenetic group B2 (46.2%), which is linked with the ExPEC pathovar, occurrence of virulence-associated genes in these strains were unexpectedly low. Due to this, and as only few of the E. coli isolates showed intermediate resistance to certain antimicrobial substances, we assume a rather naïve E. coli population, lacking contact to humans or domestic animals. Future studies featuring in depth comparative whole genome sequence analyses will provide insights into the microevolution of this interesting strain collection.

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug-resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.

INTRODUCTION:Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus. METHODS:Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on "Rodent-Borne Pathogens") in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. KR732654 and KR732653). RESULTS:The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal "hot spot" for foreign DNA integration and exchange. CONCLUSION:Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation.

Frequent contact with human waste and liquid manure from intensive livestock breeding, and the increased loads of antibiotic-resistant bacteria that result, are believed to be responsible for the high carriage rates of ESBL-producing E. coli found in birds of prey (raptors) in Central Europe. To test this hypothesis against the influence of avian migration, we initiated a comparative analysis of faecal samples from wild birds found in Saxony-Anhalt in Germany and the Gobi-Desert in Mongolia, regions of dissimilar human and livestock population characteristics and agricultural practices. We sampled a total of 281 wild birds, mostly raptors with primarily north-to-south migration routes. We determined antimicrobial resistance, focusing on ESBL production, and unravelled the phylogenetic and clonal relatedness of identified ESBL-producing E. coli isolates using multi-locus sequence typing (MLST) and macrorestriction analyses. Surprisingly, the overall carriage rates (approximately 5%) and the proportion of ESBL-producers among E. coli (Germany: 13.8%, Mongolia: 10.8%) were similar in both regions. Whereas bla(CTX-M-1) predominated among German isolates (100%), bla(CTX-M-9) was the most prevalent in Mongolian isolates (75%). We identified sequence types (STs) that are well known in human and veterinary clinical ESBL-producing E. coli (ST12, ST117, ST167, ST648) and observed clonal relatedness between a Mongolian avian ESBL-E. coli (ST167) and a clinical isolate of the same ST that originated in a hospitalised patient in Europe. Our data suggest the influence of avian migratory species in the transmission of ESBL-producing E. coli and challenge the prevailing assumption that reducing human influence alone invariably leads to lower rates of antimicrobial resistance.

Urban rats present a global public health concern as they are considered a reservoir and vector of zoonotic pathogens, including Escherichia coli. In view of the increasing emergence of antimicrobial resistant E. coli strains and the on-going discussion about environmental reservoirs, we intended to analyse whether urban rats might be a potential source of putatively zoonotic E. coli combining resistance and virulence. For that, we took fecal samples from 87 brown rats (Rattus norvegicus) and tested at least three E. coli colonies from each animal. Thirty two of these E. coli strains were pre-selected from a total of 211 non-duplicate isolates based on their phenotypic resistance to at least three antimicrobial classes, thus fulfilling the definition of multiresistance. As determined by multilocus sequence typing (MLST), these 32 strains belonged to 24 different sequence types (STs), indicating a high phylogenetic diversity. We identified STs, which frequently occur among extraintestinal pathogenic E. coli (ExPEC), such as STs 95, 131, 70, 428, and 127. Also, the detection of a number of typical virulence genes confirmed that the rats tested carried ExPEC-like strains. In particular, the finding of an Extended-spectrum beta-lactamase (ESBL)-producing strain which belongs to a highly virulent, so far mainly human- and avian-restricted ExPEC lineage (ST95), which expresses a serogroup linked with invasive strains (O18:NM:K1), and finally, which produces an ESBL-type frequently identified among human strains (CTX-M-9), pointed towards the important role, urban rats might play in the transmission of multiresistant and virulent E. coli strains. Indeed, using a chicken infection model, this strain showed a high in vivo pathogenicity. Imagining the high numbers of urban rats living worldwide, the way to the transmission of putatively zoonotic, multiresistant, and virulent strains might not be far ahead. The unforeseeable consequences of such an emerging public health threat need careful consideration in the future.