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Abstract
Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide‐mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub‐micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long‐chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo‐based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell‐based activity assay.
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.
Natural products comprise a rich reservoir for innovative drug leads and are a constant
source of bioactive compounds. To find pharmacological targets for new or already known
natural products using modern computer-aided methods is a current endeavor in drug discovery.
Nature’s treasures, however, could be used more effectively. Yet, reliable pipelines for the
large-scale target prediction of natural products are still rare. We developed an in silico workflow
Int. J. Mol. Sci. 2020, 21, 7102; doi:10.3390/ijms21197102 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020, 21, 7102 2 of 18
consisting of four independent, stand-alone target prediction tools and evaluated its performance
on dihydrochalcones (DHCs)—a well-known class of natural products. Thereby, we revealed
four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1,
17β-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a
thorough strategy on how to perform computational target predictions and guidance on using the
respective tools.
Within the last decades cancer treatment improved by the availability of more specifically
acting drugs that address molecular target structures in cancer cells. However, those target-sensitive
drugs suffer from ongoing resistances resulting from mutations and moreover they are affected
by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be
treated with the common drugs, so that there have been long efforts to develop drugs to combat
that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in
cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective
efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those
inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4
we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a
MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition
of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active
compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant
anticancer drug as proof-of-principle to encourage for further preclinical studie
The objectives of this study were to ascertain the fecal ESBL/AmpC-E. coli prevalence and to detect risk factors for their occurrence in young pre-weaned calves and their dams on large dairy farms in Germany. From 2018–2019 we investigated 2816 individual fecal samples from pre-weaned dairy calves and their dams, representing seventy-two farms (mean = 667 milking cows) from eight German federal states. To assess possible risk factors associated with ESBL/AmpC-E. coli prevalence in calves and dams, a questionnaire was performed, collecting management data. We observed an ESBL/AmpC-E. coli prevalence of 63.5% (95% CI: 57.4–69.5) among the sampled calves and 18.0% (95% CI: 12.5–23.5) among the dams. On all farms, at least one positive sample was obtained. To date, this is the highest ESBL/AmpC-E. coli prevalence observed in dairy herds in Europe. Feeding with waste milk was identified as a significant risk factor for a high prevalence of ESBL/AmpC-E. coli in calves. Many calves at large dairies in Germany are fed with waste milk due to the large amounts generated as a result of antibiotic dry-off routines and mastitis treatment with antibiotics. Other notable risk factors for high ESBL/AmpC-E. coli in calves were the general fitness/health of dams and calves, and the quality of farm hygiene. Taken together, these findings suggest that new or improved approaches to animal health management, for example, antibiotic dry cow management (selective dry cow therapy) and mastitis treatment (high self-recovery), as well as farm hygiene, should be researched and implemented.
Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations
(2021)
Transcription factors play a crucial role in regulating biological processes such as cell
growth, differentiation, organ development and cellular signaling. Within this group, proteins
equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding
transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a
variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary
glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which
serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown
to be critically important for efficient regulation of the BCL11B target genes and could therefore
represent a promising target for novel drug therapies. Here, we report the structural basis for
BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure
prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy
transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of
the single ZF domain and directly involved in forming the dimer interface. These findings not only
provide deep insight into how BCL11B acquires its active structure but also represent an important
step towards rational design or selection of potential inhibitors.
Controlling the time point and site of the release of active ingredients within the gastrointestinal tract after administration of oral delivery systems is still a challenge. In this study, the effect of the combination of small capsules (size 3) and large capsules (size 00) on the disintegration site and time was investigated using magnetic resonance imaging (MRI) in combination with a salivary tracer technique. As capsule shells, Vcaps® HPMC capsules, Vcaps® Plus HPMC capsules, gelatin and DRcaps® designed release capsules were used. The three HPMC-based capsules (Vcaps®, Vcaps® Plus and DRcaps® capsules) were tested as single capsules; furthermore, seven DUOCAP® capsule-in-capsule combinations were tested in a 10-way crossover open-label study in six healthy volunteers. The capsules contained iron oxide and hibiscus tea powder as tracers for visualization in MRI, and two different caffeine species (natural caffeine and 13C3) to follow caffeine release and absorption as measured by salivary levels. Results showed that the timing and location of disintegration in the gastrointestinal tract can be measured and differed when using different combinations of capsule shells. Increased variability among the six subjects was observed in most of the capsule combinations. The lowest variability in gastrointestinal localization of disintegration was observed for the DUOCAP® capsule-in-capsule configuration using a DRcaps® designed release capsule within a DRcaps® designed release outer capsule. In this combination, the inner DRcaps® designed release capsule always opened reliably after reaching the ileum. Thus, this combination enables targeted delivery to the distal small intestine. Among the single capsules tested, Vcaps® Plus HPMC capsules showed the fastest and most consistent disintegration.
Multi-drug resistant (MDR), gram-negative Enterobacteriaceae, such as Escherichia coli (E. coli) limit therapeutic options and increase morbidity, mortality, and treatment costs worldwide. They pose a serious burden on healthcare systems, especially in developing countries like Rwanda. Several studies have shown the effects caused by the global spread of extended-spectrum beta-lactamase (ESBL)-producing E. coli. However, limited data is available on transmission dynamics of these pathogens and the mobile elements they carry in the context of clinical and community locations in Sub-Saharan Africa. Here, we examined 120 ESBL-producing E. coli strains from patients hospitalized in the University Teaching Hospital of Butare (Rwanda), their attending caregivers as well as associated community members and livestock. Based on whole-genome analysis, the genetic diversification and phylogenetics were assessed. Moreover, the content of carried plasmids was characterized and investigated for putative transmission among strains, and for their potential role as drivers for the spread of antibiotic resistance. We show that among the 30 different sequence types (ST) detected were the pandemic clonal lineages ST131, ST648 and ST410, which combine high-level antimicrobial resistance with virulence. In addition to the frequently found resistance genes blaCTX–M–15, tet(34), and aph(6)-Id, we identified csg genes, which are required for curli fiber synthesis and thus biofilm formation. Numerous strains harbored multiple virulence-associated genes (VAGs) including pap (P fimbriae adhesion cluster), fim (type I fimbriae) and chu (Chu heme uptake system). Furthermore, we found phylogenetic relationships among strains from patients and their caregivers or related community members and animals, which indicates transmission of pathogens. Also, we demonstrated the presence and potential transfer of identical/similar ESBL-plasmids in different strains from the Rwandan setting and when compared to an external plasmid. This study highlights the circulation of clinically relevant, pathogenic ESBL-producing E. coli among patients, caregivers and the community in Rwanda. Combining antimicrobial resistance with virulence in addition to the putative exchange of mobile genetic elements among bacterial pathogens poses a significant risk around the world.
Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis was able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.
The EyeFlowCell: Development of a 3D-Printed Dissolution Test Setup for Intravitreal Dosage Forms
(2021)
An in vitro dissolution model, the so-called EyeFlowCell (EFC), was developed to test intravitreal dosage forms, simulating parameters such as the gel-like consistency of the vitreous body. The developed model consists of a stereolithography 3D-printed flow-through cell with a polyacrylamide (PAA) gel as its core. This gel needed to be coated with an agarose sheath because of its low viscosity. Drug release from hydroxypropyl methylcellulose-based implants containing either triamcinolone acetonide or fluorescein sodium was studied in the EFC using a schematic eye movement by the EyeMovementSystem (EyeMoS). For comparison, studies were performed in USP apparatus 4 and USP apparatus 7. Significantly slower drug release was observed in the PAA gel for both model drugs compared with the compendial methods. Drug release from fluorescein sodium-containing model implants was completed after 40 min in USP apparatus 4, whereas drug release in the gel-based EFC lasted 72 h. Drug release from triamcinolone acetonide-containing model implants was completed after 35 min in USP apparatus 4 and after 150 min in USP apparatus 7, whereas this was delayed until 96 h in the EFC. These results suggest that compendial release methods may overestimate the drug release rate in the human vitreous body. Using a gel-based in vitro release system such as the EFC may better predict drug release.
Previous research identified veterinary clinics as hotspots with respect to accumulation and spread of multidrug resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (EC). Therefore, promoting the prudent use of antibiotics to decrease selective pressure in that particular clinical environment is preferable to enhance biosecurity for animal patients and hospital staff. Accordingly, this study comparatively investigated the impact of two distinct perioperative antibiotic prophylaxis (PAP) regimens (short-term versus prolonged) on ESBL-EC carriage of horses subjected to colic surgery. While all horses received a combination of penicillin/gentamicin (P/G) as PAP, they were assigned to either the “single-shot group” (SSG) or the conventional “5-day group” (5DG). Fecal samples collected on arrival (t0), on the 3rd (t1) and on the 10th day after surgery (t2) were screened for ESBL-EC. All isolates were further investigated using whole genome sequences. In total, 81 of 98 horses met the inclusion criteria for this study. ESBL-EC identified in samples available at t0, t1 and t2 were 4.8% (SSG) and 9.7% (5DG), 37% (SSG) and 47.2% (5DG) as well as 55.6% (SSG) and 56.8% (5DG), respectively. Regardless of the P/G PAP regimen, horses were 9.12 times (95% CI 2.79–29.7) more likely to carry ESBL-EC at t1 compared to t0 (p < 0.001) and 15.64 times (95% CI 4.57–53.55) more likely to carry ESBL-EC at t2 compared to t0 (p < 0.001). ESBL-EC belonging to sequence type (ST) 10, ST86, ST641, and ST410 were the most prevalent lineages, with blaCTX–M–1 (60%) being the dominant ESBL gene. A close spatio-temporal relationship between isolates sharing a particular ST was revealed by genome analysis, strongly indicating local spread. Consequently, hospitalization itself has a strong impact on ESBL-EC isolation rates in horses, possibly masking differences between distinct PAP regimens. The results of this study reveal accumulation and spread of multi-drug resistant ESBL-EC among horses subjected to colic surgery with different P/G PAP regimens, challenging the local hygiene management system and work-place safety of veterinary staff. Moreover, the predominance of particular ESBL-EC lineages in clinics providing health care for horses needs further investigation.
Klebsiella pneumoniae is a common member of the intestinal flora of vertebrates. In addition to opportunistic representatives, hypervirulent (hvKp) and antibiotic-resistant K. pneumoniae (ABR-Kp) occur. While ABR-Kp isolates often cause difficult-to-treat diseases due to limited therapeutic options, hvKp is a pathotype that can infect healthy individuals often leading to recurrent infection. Here, we investigated the clinical K. pneumoniae isolate PBIO3459 obtained from a blood sample, which showed an unusual colony morphology. By combining whole-genome and RNA sequencing with multiple in vitro and in vivo virulence-associated assays, we aimed to define the respective Klebsiella subtype and explore the unusual phenotypic appearance. We demonstrate that PBIO3459 belongs to sequence type (ST)20 and carries no acquired resistance genes, consistent with phenotypic susceptibility tests. In addition, the isolate showed low-level virulence, both at genetic and phenotypic levels. We thus suggest that PBIO3459 is an opportunistic (commensal) K. pneumoniae isolate. Genomic comparison of PBIO3459 with closely related ABR-Kp ST20 isolates revealed that they differed only in resistance genes. Finally, the unusual colony morphology was mainly associated with carbohydrate and amino acid transport and metabolism. In conclusion, our study reveals the characteristics of a Klebsiella sepsis isolate and suggests that opportunistic representatives likely acquire and accumulate antibiotic resistances that subsequently enable their emergence as ABR-Kp pathogens.
Essential Oils as Multicomponent Mixtures and Their Potential for Human Health and Well-Being
(2022)
Essential oils (EOs) and their individual volatile organic constituents have been an inherent part of our civilization for thousands of years. They are widely used as fragrances in perfumes and cosmetics and contribute to a healthy diet, but also act as active ingredients of pharmaceutical products. Their antibacterial, antiviral, and anti-inflammatory properties have qualified EOs early on for both, the causal and symptomatic therapy of a number of diseases, but also for prevention. Obtained from natural, mostly plant materials, EOs constitute a typical example of a multicomponent mixture (more than one constituent substances, MOCS) with up to several hundreds of individual compounds, which in a sophisticated composition make up the property of a particular complete EO. The integrative use of EOs as MOCS will play a major role in human and veterinary medicine now and in the future and is already widely used in some cases, e.g., in aromatherapy for the treatment of psychosomatic complaints, for inhalation in the treatment of respiratory diseases, or topically administered to manage adverse skin diseases. The diversity of molecules with different functionalities exhibits a broad range of multiple physical and chemical properties, which are the base of their multi-target activity as opposed to single isolated compounds. Whether and how such a broad-spectrum effect is reflected in natural mixtures and which kind of pharmacological potential they provide will be considered in the context of ONE Health in more detail in this review.
The present study covers the synthesis, purification and evaluation of a novel aminomethacrylate-based copolymer in terms of its suitability for improving the solubility and in vitro release of poorly water-soluble drug compounds. The new copolymer was synthesized by solvent polymerization with radical initiation and by use of a chain transfer agent. Based on its composition, it can be considered as a modified type of dimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylate “EUDRAGIT® E PO” (ModE). ModE was specifically developed to provide a copolymer with processing and application properties that exceed those of commercially available (co-)polymers in solubility enhancement technologies where possible. By varying the concentration of the chain transfer agent in the radical polymerization process, the molecular weight of ModE was varied in a range of 173–305 kDa. To evaluate the solubility-enhancing properties of ModE, a series of drug-loaded extrudates were prepared by hot melt extrusion using the novel—as well as several commercially available—(co-)polymers. These extrudates were then subjected to comparative tests for amorphousness, solubility-enhancing properties, storage stability, and drug release. Celecoxib, efavirenz, and fenofibrate were used as model drugs in all experiments. Of all the (co-)polymers included in the study, ModE with a molecular weight of 173 kDa showed the best performance in terms of desired properties and was shown to be particularly suitable for preparing amorphous solid dispersions (ASDs) of the three model drugs, which in a first set of dissolution experiments showed better release behavior under pH conditions of the fasting stomach than higher molecular weight ModE types, as well as a variety of commercially available (co-)polymers. Therefore, the results demonstrate the successful synthesis of a new copolymer, which in future studies will be investigated in more detail for universal application in the field of solubility enhancement.
Antimicrobial resistance is an increasing global problem and complicates successful treatments of bacterial infections in animals and humans. We conducted a longitudinal study in Mecklenburg-Western Pomerania to compare the occurrence of ESBL-producing Escherichia (E.) coli in three conventional and four organic pig farms. ESBL-positive E. coli, especially of the CTX-M type, were found in all fattening farms, confirming that antimicrobial resistance is widespread in pig fattening and affects both conventional and organic farms. The percentage of ESBL-positive pens was significantly higher on conventional (55.2%) than on organic farms (44.8%) with similar proportions of ESBL-positive pens on conventional farms (54.3–61.9%) and a wide variation (7.7–84.2%) on organic farms. Metadata suggest that the farms of origin, from which weaner pigs were purchased, had a major influence on the occurrence of ESBL-producing E. coli in the fattening farms. Resistance screening showed that the proportion of pens with multidrug-resistant E. coli was similar on conventional (28.6%) and organic (31.5%) farms. The study shows that ESBL-positive E. coli play a major role in pig production and that urgent action is needed to prevent their spread.
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.
Fibers and yarns are part of everyday life. So far, fibers that are also used pharmaceutically have mainly been produced by electrospinning. The common use of spinning oils and the excipients they contain, in connection with production by melt extrusion, poses a regulatory challenge for pharmaceutically usable fibers. In this publication, a newly developed small-scale direct-spinning melt extrusion system is described, and the pharmaceutically useful polyvinyl filaments produced with it are characterized. The major parts of the system were newly developed or extensively modified and manufactured cost-effectively within a short time using rapid prototyping (3D printing) from various materials. For example, a stainless-steel spinneret was developed in a splice design for a table-top melt extrusion system that can be used in the pharmaceutical industry. The direct processing of the extruded fibers was made possible by a spinning system developed called Spinning-Rosi, which operates continuously and directly in the extrusion process and eliminates the need for spinning oils. In order to prevent instabilities in the product, further modifications were also made to the process, such as a the moisture encapsulation of the melt extrusion line at certain points, which resulted in a bubble-free extrudate with high tensile strength, even in a melt extrusion line without built-in venting.
The study aimed to examine the influence of a rotating magnetic field (RMF) of two different frequencies (5 and 50 Hz) on the expression of regulatory (agrA, hld, rot) and staphylococcal enterotoxin (SE—sea, sec, sel) genes as well as the production of SEs (SEA, SEC, SEL) by the Staphylococcus aureus FRI913 strain cultured on a medium supplemented with a subinhibitory concentration of trans-anethole (TA). Furthermore, a theoretical model of interactions between the bacterial medium and bacterial cells exposed to RMF was proposed. Gene expression and SEs production were measured using quantitative real-time PCR and ELISA techniques, respectively. Based on the obtained results, it was found that there were no significant differences in the expression of regulatory and SE genes in bacteria simultaneously cultured on a medium supplemented with TA and exposed to RMF at the same time in comparison to the control (unexposed to TA and RMF). In contrast, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF or when they were exposed to RMF of 50 Hz (but not to TA), a significant increase in agrA and sea transcripts as compared to the unexposed control was found. Moreover, the decreased level of sec transcripts in bacteria cultured without TA but exposed to RMF of 50 Hz was also revealed. In turn, a significant increase in SEA and decrease in SEC and SEL production was observed in bacteria cultured on a medium supplemented with TA and simultaneously exposed to RMFs. It can be concluded, that depending on SE and regulatory genes expression as well as production of SEs, the effect exerted by the RMF and TA may be positive (i.e., manifests as the increase in SEs and/or regulatory gene expression of SEs production) or negative (i.e., manifests as the reduction in both aforementioned features) or none.
Application of In Vivo Imaging Techniques and Diagnostic Tools in Oral Drug Delivery Research
(2022)
Drug absorption following oral administration is determined by complex and dynamic interactions between gastrointestinal (GI) physiology, the drug, and its formulation. Since many of these interactions are not fully understood, the COST action on “Understanding Gastrointestinal Absorption-related Processes (UNGAP)” was initiated in 2017, with the aim to improve the current comprehension of intestinal drug absorption and foster future developments in this field. In this regard, in vivo techniques used for the characterization of human GI physiology and the intraluminal behavior of orally administered dosage forms in the GI tract are fundamental to gaining deeper mechanistic understanding of the interplay between human GI physiology and drug product performance. In this review, the potential applications, advantages, and limitations of the most important in vivo techniques relevant to oral biopharmaceutics are presented from the perspectives of different research fields.
Highly Virulent and Multidrug-Resistant Escherichia coli Sequence Type 58 from a Sausage in Germany
(2022)
Studies have previously described the occurrence of multidrug-resistant (MDR) Escherichia coli in human and veterinary medical settings, livestock, and, to a lesser extent, in the environment and food. While they mostly analyzed foodborne E. coli regarding phenotypic and sometimes genotypic antibiotic resistance and basic phylogenetic classification, we have limited understanding of the in vitro and in vivo virulence characteristics and global phylogenetic contexts of these bacteria. Here, we investigated in-depth an E. coli strain (PBIO3502) isolated from a pork sausage in Germany in 2021. Whole-genome sequence analysis revealed sequence type (ST)58, which has an internationally emerging high-risk clonal lineage. In addition to its MDR phenotype that mostly matched the genotype, PBIO3502 demonstrated pronounced virulence features, including in vitro biofilm formation, siderophore secretion, serum resilience, and in vivo mortality in Galleria mellonella larvae. Along with the genomic analysis indicating close phylogenetic relatedness of our strain with publicly available, clinically relevant representatives of the same ST, these results suggest the zoonotic and pathogenic character of PBIO3502 with the potential to cause infection in humans and animals. Additionally, our study highlights the necessity of the One Health approach while integrating human, animal, and environmental health, as well as the role of meat products and food chains in the putative transmission of MDR pathogens.
Antimicrobial resistance (AMR) is a serious global health threat and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are a major contributor. This study aimed to gain a deeper insight into the AMR burden of wild animals. In total, 1595 fecal samples were collected by two systematic searches in Mecklenburg-Western Pomerania, north-east Germany. Samples were screened for ESBL-carrying Escherichia (E.) coli and isolates found were further analyzed using antimicrobial susceptibility testing and whole-genome sequencing. We found an estimated prevalence of 1.2% ESBL-producing E. coli in wild boar and 1.1% in wild ruminants. CTX-M-1 was the most abundant CTX-M type. We also examined fecal samples from wild boar and wild ruminants using shotgun metagenomics to gain insight into the resistome in wild animals. The latter revealed significantly lower normalized counts for AMR genes in wildlife samples compared to farm animals. The AMR gene levels were lower in wild ruminants than in wild boar. In conclusion, our study revealed a low prevalence of ESBL-producing E. coli and a low overall AMR gene burden in wild boar and wild ruminants, probably due to the secluded location of the search area.
Characterization, Chemical Compounds and Biological Activities of Marrubium vulgare L. Essential Oil
(2022)
As consumer trends shift towards more natural and ecological consumption patterns, industrialists are actively working towards substituting synthetic chemicals with natural and vegan products that contain bioactive properties. Thus, considering the shifts in customer demand and the growing concern around vegetable sourced productions, this work aims to contribute to the valorization of aromatic and medicinal Moroccan plants. By focusing on the Marrubium vulgare L. species, our objective is to carry out a physicochemical characterization to determine its chemical composition and biological activities. The volatile fraction collected by hydrodistillation (0.61%) and analyzed by GC-MS (gas chromatography coupled to mass spectrometry) contains five main compounds: 3-Thujanone, Eugenol, Topanol, Menthone and Piperitone. The antioxidant activity has been estimated by applying the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test and the ferric reducing antioxidant power (FRAP). The values of inhibitory concentration prove that our oil is a good antioxidant, with values of IC50 = 1.136 mg/mL and IC50 = 2.998 mg/mL, respectively, for the DPPH and FRAP tests. The results of the antifungal activity indicate a significant inhibition of mycelial growth for both tested molds, as well as a total inhibition of spore production at a concentration of 0.25 µL/mL.
Abstract
Aim
To verify synergistic effects, we investigated the antimicrobial activity of seven phenolic phytochemicals (gallic acid; epicatechin; epigallocatechin gallate; daidzein; genistein; myricetin; 3‐hydroxy‐6‐methoxyflavone) in combination with six antibiotics against multidrug‐resistant isolates from the ESKAPE group.
Methods and Results
To investigate single phytochemicals and combinations, initial microdilution and checkerboard assays were used, followed by time‐kill assays to evaluate the obtained results. The research revealed that phenolic compounds on their own resulted in little or no inhibitory effects. During preliminary tests, most of the combinations resulted in indifference (134 [71.3%]). In all, 30 combinations led to antagonism (15.9%); however, 24 showed synergistic effects (12.8%). The main tests resulted in nine synergistic combinations for the treatment of four different bacteria strains, including two substances (3‐hydroxy‐6‐methoxyflavone, genistein) never tested before in such setup. Time‐kill curves for combinations with possible synergistic effects confirmed the results against Acinetobacter baumannii as the one with the greatest need for research.
Conclusions
The results highlight the potential use of antibiotic–phytocompound combinations for combating infections with multi‐resistant pathogens. Synergistic combinations could downregulate the resistance mechanisms of bacteria.
Significance and Impact of the Study
The aim of this study is to demonstrate the potential use of phenolic natural compounds in combination with conventional antibiotics against multidrug‐resistant bacteria of the ESKAPE group. Due to synergistic effects of natural phenolic compounds combined with antibiotics, pathogens that are already resistant to antibiotics could be resensitized as we were able to reduce their MICs back to sensitive. In addition, combination therapies could prevent the development of resistance by reducing the dose of antibiotics. This approach opens up the basis for future development of antimicrobial therapy strategies, which are so urgently needed in the age of multidrug‐resistant pathogens.
Livestock animals, especially poultry, are a known reservoir for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli). They may enter the pen either via positive day-old chicks or via the environment. We developed a mathematical model to illustrate the entry and dissemination of resistant bacteria in a broiler pen during one fattening period in order to investigate the effectiveness of intervention measures on this infection process. Different management measures, such as varying amounts of litter, a slow-growing breed or lower stocking densities, were tested for their effects on broiler colonization. We also calculated the impact of products that may influence the microbiota in the chicks’ digestive tract, such as pre- or probiotics, feed supplements or competitive exclusion products. Our model outcomes show that a contaminated pen or positive chicks at the beginning of the fattening period can infect the entire flock. Increasing the amount of litter and decreasing the stocking density were shown to be effective in our model. Differences in the route of entry were found: if the chicks are already positive, the litter quantity must be increased to at least six times the standard of 1000 g/m2, whereas, if the pen is contaminated on the first day, three times the litter quantity is sufficient. A reduced stocking density of 20 kg/m2 had a significant effect on the incidence of infection only in a previously contaminated pen. Combinations of two or three measures were effective in both scenarios; similarly, feed additives may be beneficial in reducing the growth rate of ESBL-producing E. coli. This model is a valuable tool for evaluating interventions to reduce the transmission and spread of resistant bacteria in broiler houses. However, data are still needed to optimize the model, such as growth rates or survival data of ESBL-producing E. coli in different environments.
The present study focused on a new formulation approach to improving the solubility of drugs with poor aqueous solubility. A hot melt extrusion (HME) process was applied to prepare drug-loaded solid self-nanoemulsifying drug delivery systems (S-SNEDDS) by co-extrusion of liquid SNEDDS (L-SNEDDS) and different polymeric carriers. Experiments were performed with L-SNEDDS formulations containing celecoxib, efavirenz or fenofibrate as model drugs. A major objective was to identify a polymeric carrier and process parameters that would enable the preparation of stable S-SNEDDS without impairing the release behavior and storage stability of the L-SNEDDS used and, if possible, even improving them further. In addition to commercially available (co)polymers already used in the field of HME, a particular focus was on the evaluation of different variants of a recently developed aminomethacrylate-based copolymer (ModE) that differed in Mw. Immediately after preparation, the L-SNEDDS and S-SNEDDS formulations were tested for amorphicity by differential scanning calorimetry. Furthermore, solubility and dissolution tests were performed. In addition, the storage stability was investigated at 30 °C/65% RH over a period of three and six months, respectively. In all cases, amorphous formulations were obtained and, especially for the model drug celecoxib, S-SNEDDS were developed that maintained the rapid and complete drug release of the underlying L-SNEDDS even over an extended storage period. Overall, the data obtained in this study suggest that the presented S-SNEDDS approach is very promising, provided that drug-loaded L-SNEDDS are co-processed with a suitable polymeric carrier. In the case of celecoxib, the E-173 variant of the novel ModE copolymer proved to be a novel polymeric carrier with great potential for application in S-SNEDDS. The presented approach will, therefore, be pursued in future studies to establish S-SNEDDS as an alternative formulation to other amorphous systems.