De novo genome assembly resolving repetitive structures enables genomic analysis of 35 European Mycoplasmopsis bovis strains.

Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.

Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Inclusion of Oxford Nanopore long reads improves all microbial and viral metagenome-assembled genomes from a complex aquifer system.

Assembling microbial and viral genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. To compare the recovery of genomes from microorganisms and their viruses from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore Technologies (ONT) sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from an Illumina-only assembly, ONT-only assembly, and a hybrid assembly approach. The hybrid approach recovered 2× more mid to high-quality MAGs compared to the Illumina-only approach and 4× more than the ONT-only approach. A similar number of viral genomes were reconstructed using the hybrid and ONT methods, and both recovered nearly fourfold more viral genomes than the Illumina-only approach. While yielding fewer MAGs, the ONT-only approach generated MAGs with a high probability of containing rRNA genes, 3× higher than either of the other methods. Of the shared MAGs recovered from each method, the ONT-only approach generated the longest and least fragmented MAGs, while the hybrid approach yielded the most complete. This work provides quantitative data to inform a cost-benefit analysis of the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

Cry1Ac production is costly for native plants attacked by non-Cry1Ac-targeted herbivores in the field.

Plants are the primary producers in most terrestrial ecosystems and have complex defense systems to protect their produce. Defense-deficient, high-yielding agricultural monocultures attract abundant nonhuman consumers, but are alternatively defended through pesticide application and genetic engineering to produce insecticidal proteins such as Cry1Ac (Bacillus thuringiensis). These approaches alter the balance between yield protection and maximization but have been poorly contextualized to known yield-defense trade-offs in wild plants. The native plant Nicotiana attenuata was used to compare yield benefits of plants transformed to be defenseless to those with a full suite of naturally evolved defenses, or additionally transformed to ectopically produce Cry1Ac. An insecticide treatment allowed us to examine yield under different herbivore loads in N. attenuata's native habitat. Cry1Ac, herbivore damage, and growth parameters were monitored throughout the season. Biomass and reproductive correlates were measured at season end. Non-Cry1Ac-targeted herbivores dominated on noninsecticide-treated plants, and increased the yield drag of Cry1Ac-producing plants in comparison with endogenously defended or undefended plants. Insecticide-sprayed Cry1Ac-producing plants lagged less in stalk height, shoot biomass, and flower production. In direct comparison with the endogenous defenses of a native plant, Cry1Ac production did not provide yield benefits for plants under observed herbivore loads in a field study.

Trichobaris weevils distinguish amongst toxic host plants by sensing volatiles that do not affect larval performance.

Herbivorous insects use plant metabolites to inform their host plant selection for oviposition. These host-selection behaviours are often consistent with the preference-performance hypothesis; females oviposit on hosts that maximize the performance of their offspring. However, the metabolites used for these oviposition choices and those responsible for differences in offspring performance remain unknown for ecologically relevant interactions. Here, we examined the host-selection behaviours of two sympatric weevils, the Datura (Trichobaris compacta) and tobacco (T. mucorea) weevils in field and glasshouse experiments with transgenic host plants specifically altered in different components of their secondary metabolism. Adult females of both species strongly preferred to feed on D. wrightii rather than on N. attenuata leaves, but T. mucorea preferred to oviposit on N. attenuata, while T. compacta oviposited only on D. wrightii. These oviposition behaviours increased offspring performance: T. compacta larvae only survived in D. wrightii stems and T. mucorea larvae survived better in N. attenuata than in D. wrightii stems. Choice assays with nicotine-free, JA-impaired, and sesquiterpene-over-produced isogenic N. attenuata plants revealed that although half of the T. compacta larvae survived in nicotine-free N. attenuata lines, nicotine did not influence the oviposition behaviours of both the nicotine-adapted and nicotine-sensitive species. JA-induced sesquiterpene volatiles are key compounds influencing T. mucorea females' oviposition choices, but these sesquiterpenes had no effect on larval performance. We conclude that adult females are able to choose the best host plant for their offspring and use chemicals different from those that influence larval performance to inform their oviposition decisions.

From Shadows to Spotlight: Enhancing Bacterial DNA Detection in Blood Samples through Cutting-Edge Molecular Pre-Amplification.

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium. A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae, respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.

Molecular Characterization of Chimeric Staphylococcus aureus Strains from Waterfowl.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

Characterisation of PVL-Positive Staphylococcus argenteus from the United Arab Emirates.

Staphylococcus argenteus is a recently described staphylococcal species that is related to Staphylococcus aureus but lacks the staphyloxanthin operon. It is able to acquire both resistance markers such as the SCCmec elements and mobile genetic elements carrying virulence-associated genes from S. aureus. This includes those encoding the Panton-Valentine leukocidin (PVL), which is associated mainly with severe and/or recurrent staphylococcal skin and soft tissue infections. Here, we describe the genome sequences of two PVL-positive, mecA-negative S. argenteus sequence type (ST) 2250 isolates from the United Arab Emirates in detail. The isolates were found in a dental clinic in the United Arab Emirates (UAE). Both were sequenced using Oxford Nanopore Technology (ONT). This demonstrated the presence of temperate bacteriophages in the staphylococcal genomes, including a PVL prophage. It was essentially identical to the published sequence of phiSa2wa_st78 (GenBank NC_055048), a PVL phage from an Australian S. aureus clonal complex (CC) 88 isolate. Besides the PVL prophage, one isolate carried another prophage and the second isolate carried two additional prophages, whereby the region between these two prophages was inverted. This "flipped" region comprised about 1,083,000 bp, or more than a third of the strain's genome, and it included the PVL prophage. Prophages were induced by Mitomycin C treatment and subjected to transmission electron microscopy (TEM). This yielded, in accordance to the sequencing results, one or, respectively, two distinct populations of icosahedral phages. It also showed prolate phages which presumptively might be identified as the PVL phage. This observation highlights the significance bacteriophages have as agents of horizontal gene transfer as well as the need for monitoring emerging staphylococcal strains, especially in cosmopolitan settings such as the UAE.

Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata.

Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.

Analyses of wrky18 wrky40 plants reveal critical roles of SA/EDS1 signaling and indole-glucosinolate biosynthesis for Golovinomyces orontii resistance and a loss-of resistance towards Pseudomonas syringae pv. tomato AvrRPS4.

Simultaneous mutation of two WRKY-type transcription factors, WRKY18 and WRKY40, renders otherwise susceptible wild-type Arabidopsis plants resistant towards the biotrophic powdery mildew fungus Golovinomyces orontii. Resistance in wrky18 wrky40 double mutant plants is accompanied by massive transcriptional reprogramming, imbalance in salicylic acid (SA) and jasmonic acid (JA) signaling, altered ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) expression, and accumulation of the phytoalexin camalexin. Genetic analyses identified SA biosynthesis and EDS1 signaling as well as biosynthesis of the indole-glucosinolate 4MI3G as essential components required for loss-of-WRKY18 WRKY40-mediated resistance towards G. orontii. The analysis of wrky18 wrky40 pad3 mutant plants impaired in camalexin biosynthesis revealed an uncoupling of pre- from postinvasive resistance against G. orontii. Comprehensive infection studies demonstrated the specificity of wrky18 wrky40-mediated G. orontii resistance. Interestingly, WRKY18 and WRKY40 act as positive regulators in effector-triggered immunity, as the wrky18 wrky40 double mutant was found to be strongly susceptible towards the bacterial pathogen Pseudomonas syringae DC3000 expressing the effector AvrRPS4 but not against other tested Pseudomonas strains. We hypothesize that G. orontii depends on the function of WRKY18 and WRKY40 to successfully infect Arabidopsis wild-type plants while, in the interaction with P. syringae AvrRPS4, they are required to mediate effector-triggered immunity.

Petunia flowers solve the defence/apparency dilemma of pollinator attraction by deploying complex floral blends.

Flowers recruit floral visitors for pollination services by emitting fragrances. These scent signals can be intercepted by antagonists such as florivores to locate host plants. Hence, as a consequence of interactions with both mutualists and antagonists, floral bouquets likely consist of both attractive and defensive components. While the attractive functions of floral bouquets have been studied, their defensive function has not, and field-based evidence for the deterrence of floral-scent constituents is lacking. In field and glasshouse experiments with five lines of transgenic Petunia x hybrida plants specifically silenced in their ability to release particular components of their floral volatile bouquet, we demonstrate that the emission of single floral-scent compounds can dramatically decrease damage from generalist florivores. While some compounds are used in host location, others prevent florivory. We conclude that the complex blends that comprise floral scents are likely sculpted by the selective pressures of both pollinators and herbivores.

Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection.

The Arabidopsis (Arabidopsis thaliana) transcription factor WRKY33 is essential for defense toward the necrotrophic fungus Botrytis cinerea. Here, we aimed at identifying early transcriptional responses mediated by WRKY33. Global expression profiling on susceptible wrky33 and resistant wild-type plants uncovered massive differential transcriptional reprogramming upon B. cinerea infection. Subsequent detailed kinetic analyses revealed that loss of WRKY33 function results in inappropriate activation of the salicylic acid (SA)-related host response and elevated SA levels post infection and in the down-regulation of jasmonic acid (JA)-associated responses at later stages. This down-regulation appears to involve direct activation of several jasmonate ZIM-domain genes, encoding repressors of the JA-response pathway, by loss of WRKY33 function and by additional SA-dependent WRKY factors. Moreover, genes involved in redox homeostasis, SA signaling, ethylene-JA-mediated cross-communication, and camalexin biosynthesis were identified as direct targets of WRKY33. Genetic studies indicate that although SA-mediated repression of the JA pathway may contribute to the susceptibility of wrky33 plants to B. cinerea, it is insufficient for WRKY33-mediated resistance. Thus, WRKY33 apparently directly targets other still unidentified components that are also critical for establishing full resistance toward this necrotroph.

Characterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt.

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt's second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.

Characterisation of a Staphylococcus aureus Isolate Carrying Phage-Borne Enterotoxin E from a European Badger (Meles meles).

Staphylococcus (S.) aureus colonizes up to 30% of all humans and can occasionally cause serious infections. It is not restricted to humans as it can also often be found in livestock and wildlife. Recent studies have shown that wildlife strains of S. aureus usually belong to other clonal complexes than human strains and that they might differ significantly with regard to the prevalence of genes encoding antimicrobial resistance properties and virulence factors. Here, we describe a strain of S. aureus isolated from a European badger (Meles meles). For molecular characterisation, DNA microarray-based technology was combined with various next-generation sequencing (NGS) methods. Bacteriophages from this isolate were induced with Mitomycin C and characterized in detail by transmission electron microscopy (TEM) and NGS. The S. aureus isolate belonged to ST425 and had a novel spa repeat sequence (t20845). It did not carry any resistance genes. The uncommon enterotoxin gene see was detected in one of its three temperate bacteriophages. It was possible to demonstrate the induction of all three prophages, although only one of them was expected to be capable of excision based on its carriage of the excisionase gene xis. All three bacteriophages belonged to the family Siphoviridae. Minor differences in size and shape of their heads were noted in TEM images. The results highlight the ability of S. aureus to colonize or infect different host species successfully, which can be attributed to a variety of virulence factors on mobile genetic elements, such as bacteriophages. As shown in the strain described herein, temperate bacteriophages not only contribute to the fitness of their staphylococcal host by transferring virulence factors, but also increase mobility among themselves by sharing genes for excision and mobilization with other prophages.

Clonal Complexes Distribution of Staphylococcus aureus Isolates from Clinical Samples from the Caribbean Islands.

The aim of this study was to comprehensively characterise S. aureus from the Caribbean Islands of Trinidad and Tobago, and Jamaica. A total of 101 S. aureus/argenteus isolates were collected in 2020, mainly from patients with skin and soft tissue infections. They were characterised by DNA microarray allowing the detection of ca. 170 target genes and assignment to clonal complexes (CC)s and strains. In addition, the in vitro production of Panton-Valentine leukocidin (PVL) was examined by an experimental lateral flow assay. Two isolates were identified as S. argenteus, CC2596. The remaining S. aureus isolates were assigned to 21 CCs. The PVL rate among methicillin-susceptible S. aureus (MSSA) isolates was high (38/101), and 37 of the 38 genotypically positive isolates also yielded positive lateral flow results. The isolate that did not produce PVL was genome-sequenced, and it was shown to have a frameshift mutation in agrC. The high rate of PVL genes can be attributed to the presence of a known local CC8-MSSA clone in Trinidad and Tobago (n = 12) and to CC152-MSSA (n = 15). In contrast to earlier surveys, the USA300 clone was not found, although one MSSA isolate carried the ACME element, probably being a mecA-deficient derivative of this strain. Ten isolates, all from Trinidad and Tobago, were identified as MRSA. The pandemic ST239-MRSA-III strain was still common (n = 7), but five isolates showed a composite SCCmec element not observed elsewhere. Three isolates were sequenced. That showed a group of genes (among others, speG, crzC, and ccrA/B-4) to be linked to its SCC element, as previously found in some CC5- and CC8-MRSA, as well as in S. epidermidis. The other three MRSA belonged to CC22, CC72, and CC88, indicating epidemiological connections to Africa and the Middle East.

Pithy protection: Nicotiana attenuata's jasmonic acid-mediated defenses are required to resist stem-boring weevil larvae.

Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C(6) aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant's apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata's fitness.

Description of Staphylococcal Strains from Straw-Coloured Fruit Bat (Eidolon helvum) and Diamond Firetail (Stagonopleura guttata) and a Review of their Phylogenetic Relationships to Other Staphylococci.

The phylogenetic tree of the Staphylococcus aureus complex consists of several distinct clades and the majority of human and veterinary S. aureus isolates form one large clade. In addition, two divergent clades have recently been described as separate species. One was named Staphylococcus argenteus, due to the lack of the "golden" pigment staphyloxanthin. The second one is S. schweitzeri, found in humans and animals from Central and West Africa. In late 2021, two additional species, S. roterodami and S. singaporensis, have been described from clinical samples from Southeast Asia. In the present study, isolates and their genome sequences from wild Straw-coloured fruit bats (Eidolon helvum) and a Diamond firetail (Stagonopleura guttata, an estrildid finch) kept in a German aviary are described. The isolates possessed staphyloxanthin genes and were closer related to S. argenteus and S. schweitzeri than to S. aureus. Phylogenetic analysis revealed that they were nearly identical to both, S. roterodami and S. singaporensis. We propose considering the study isolates, the recently described S. roterodami and S. singaporensis as well as some Chinese strains with MLST profiles stored in the PubMLST database as different clonal complexes within one new species. According to the principle of priority we propose it should be named S. roterodami. This species is more widespread than previously believed, being observed in West Africa, Southeast Asia and Southern China. It has a zoonotic connection to bats and has been shown to be capable of causing skin and soft tissue infections in humans. It is positive for staphyloxanthin, and it could be mis-identified as S. aureus (or S. argenteus) using routine procedures. However, it can be identified based on distinct MLST alleles, and "S. aureus" sequence types ST2470, ST3135, ST3952, ST3960, ST3961, ST3963, ST3965, ST3980, ST4014, ST4075, ST4076, ST4185, ST4326, ST4569, ST6105, ST6106, ST6107, ST6108, ST6109, ST6999 and ST7342 belong to this species.

Occurrence and Characteristics of ESBL- and Carbapenemase- Producing Escherichia coli from Wild and Feral Birds in Greece.

Wild and feral birds are known to be involved in the maintenance and dissemination of clinically-important antimicrobial-resistant pathogens, such as extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae. The aim of our study was to evaluate the presence of ESBL- and carbapenemase-producing Escherichia coli among wild and feral birds from Greece and to describe their antimicrobial resistance characteristics. In this context, fecal samples of 362 birds were collected and cultured. Subsequently, the antimicrobial resistance pheno- and geno-type of all the obtained E. coli isolates were determined. A total of 12 multidrug-resistant (MDR), ESBL-producing E. coli were recovered from eight different wild bird species. Eleven of these isolates carried a blaCTX-M-1 group gene alone or in combination with blaTEM and one carried only blaTEM. AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring blaNDM along with a combination of additional resistance genes. This report describes the occurrence of ESBL- and carbapenemase-producing E. coli among wild avian species in Greece, emphasizing the importance of incorporating wild birds in the assessment of AMR circulation in non-clinical settings.

Cross-Sectional Survey of Antibiotic Resistance in Extended Spectrum ß-Lactamase-Producing Enterobacteriaceae Isolated from Pigs in Greece.

This study aimed to estimate the prevalence of extended-spectrum ß-lactamase-producing (ESBL) bacteria in swine. Thus, 214 fecal samples were collected from suckling and weaned piglets from 34 farms in Greece (out of an overall population of about 14,300 sows). A subset of 78 (36.5%) ESBL producers were identified as E. coli (69/78, 88.5%), K. pneumoniae spp. pneumoniae (3.8%), P. mirabilis (5.1%), E. cloacae complex (1.3%) and S. enterica spp. diarizonae (1.3%). Resistance to at least one class of non-ß-lactam antibiotics was detected in 78 isolates. Among the E. coli strains, resistance was identified with regard to aminoglycosides (n = 31), fluoroquinolones (n = 49), tetracycline (n = 26) and trimethoprim/sulfamethoxazole (n = 46). Of the three K. pneumoniae spp. pneumoniae, two displayed resistances to aminoglycosides and all were resistant to fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole. As for the four P. mirabilis isolates, three had a resistant phenotype for aminoglycosides and all were resistant to imipenem, fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole. Molecular characterization of the isolates revealed the presence of CTX-M, SHV and TEM genes, as well as of genes conferring resistance to fluoroquinolones, aminoglycosides, sulfonamides, trimethoprim, macrolides and colistin. High levels of antimicrobial resistance (AMR) were demonstrated in Greek swine herds posing a concern for the efficacy of treatments at the farm level as well as for public health.

Small Extracellular Vesicles from Peripheral Blood of Aged Mice Pass the Blood-Brain Barrier and Induce Glial Cell Activation.

Extracellular vesicles (EVs), including small EVs (sEVs), are involved in neuroinflammation and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Yet, increased neuroinflammation can also be detected in the aging brain, and it is associated with increased glial activation. Changes in EV concentration are reported in aging tissues and senescence cells, suggesting a role of EVs in the process of aging. Here, we investigated the effect of peripheral sEVs from aged animals on neuroinflammation, specifically on glial activation. sEVs were isolated from the peripheral blood of young (3 months) and aged (24 months) C57BL/6J wildtype mice and injected into the peripheral blood from young animals via vein tail injections. The localization of EVs and the expression of selected genes involved in glial cell activation, including Gfap, Tgf-ß, Cd68, and Iba1, were assessed in brain tissue 30 min, 4 h, and 24 h after injection. We found that sEVs from peripheral blood of aged mice but not from young mice altered gene expression in the brains of young animals. In particular, the expression of the specific astrocyte marker, Gfap, was significantly increased, indicating a strong response of this glial cell type. Our study shows that sEVs from aged mice can pass the blood-brain barrier (BBB) and induce glial cell activation.