Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022

Whole genome sequencing data of 874 Escherichia coli isolates carrying blaNDM-5 from 13 European Union/ European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying blaNDM-5 would leave limited treatment options for serious E. coli infections

Treponema spp. in necrotic skin ulcers in pigs

Treponema belongs to the phylum Spirochaetes consisting of bacteria distinguishable by a unique cell architecture that enables corkscrew-like motility. In two case studies, treponemes were isolated from ear necrosis and shoulder ulcers, two types of skin lesions that have impact on animal welfare and may cause economic losses. This doctoral thesis focuses on a hypothesis that Treponema spp. have a pathogenic role in the progression of ear necrosis and shoulder ulcers in pigs and, as Treponema spp. are present in gingiva of pigs, that transmission to skin is mediated by biting or licking. The thesis describes their occurrence, phenotypic and genetic features. We sampled 109 pigs with lesions and 60 apparently healthy piglets. Spirochetes were present in 73% of the shoulder ulcers, in 53% of the ear necroses and in 9.7% of the gingivae. Many identified phylotypes were similar to Treponema spp. considered as pathogens in bovine digital dermatitis, a claw disease in cattle. Similar phylotypes were present both in gingiva and ulcers. There were indications of transmission between gingiva and ulcers. Twelve isolates were acquired and identified as T. pedis, T. parvum and T. sp. OMZ840-like. Metabolic patterns were similar to those of other treponemes and were not discriminatory between isolates. All except two gingival isolates showed unique DNA fingerprints. Treponema sp. OMZ840-like and T. pedis isolates were hemolytic, T. parvum was not. The isolates were generally susceptible to the tested antimicrobials. The genome sequence of T. pedis strain T A4 from ear necrosis was de novo assembled and analyzed. The genome was most similar to that of T. denticola, a species strongly associated with human periodontitis. Several of the predicted genes in T. pedis were homologous to virulence related genes in T. denticola, including those encoding the major surface sheath protein, dentilisin and dentipain. In T. denticola, IdeT is a protein that includes the oligopeptidase domain dentipain. An IdeT-homologue in T. pedis strain T A4, TPE0673, was recombinantly expressed in Escherichia coli and purified for in vitro testing. TPE0673 did not display protease activity towards immunoglobulin G (IgG) or insulin as its homologues. However, western blot analysis showed that IgG in serum from sows with shoulder ulcers bound to purified TPE0673, suggesting an immunogenic property of this gene. In conclusion, this thesis describes an association between Treponema spp. with ear necrosis and shoulder ulcers. The treponemes present in pig ulcers were phylogenetically similar to those of oral origin and transmission between gingiva and ulcer was indicated. Finally, analysis of the genome of T. pedis revealed the presence of several putative virulence genes indicating a pathogenic potential for this species

Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins.

Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway

Genome-Wide Relatedness of <i>Treponema pedis,</i> from Gingiva and Necrotic Skin Lesions of Pigs, with the Human Oral Pathogen <i>Treponema denticola</i>

<div><p><i>Treponema pedis</i> and <i>T. denticola</i> are two genetically related species with different origins of isolation. <i>Treponema denticola</i> is part of the human oral microbiota and is associated with periodontitis while <i>T. pedis</i> has been isolated from skin lesions in animals, <i>e.g.,</i> digital dermatitis in cattle and necrotic ulcers in pigs. Although multiple <i>Treponema</i> phylotypes may exist in ulcerative lesions in pigs, <i>T. pedis</i> appears to be a predominant spirochete in these lesions. <i>Treponema pedis</i> can also be present in pig gingiva. In this study, we determined the complete genome sequence of <i>T. pedis</i> strain T A4, isolated from a porcine necrotic ear lesion, and compared its genome with that of <i>T. denticola</i>. Most genes in <i>T. pedis</i> were homologous to those in <i>T. denticola</i> and the two species were similar in general genomic features such as size, G+C content, and number of genes. In addition, many homologues of specific virulence-related genes in <i>T. denticola</i> were found in <i>T. pedis</i>. Comparing a selected pair of strains will usually not give a complete picture of the relatedness between two species. We therefore complemented the analysis with draft genomes from six <i>T. pedis</i> isolates, originating from gingiva and necrotic ulcers in pigs, and from twelve <i>T. denticola</i> strains. Each strain carried a considerable amount of accessory genetic material, of which a large part was strain specific. There was also extensive sequence variability in putative virulence-related genes between strains belonging to the same species. Signs of lateral gene-transfer events from bacteria known to colonize oral environments were found. This suggests that the oral cavity is an important habitat for <i>T. pedis</i>. In summary, we found extensive genomic similarities between <i>T. pedis</i> and <i>T. denticola</i> but also large variability within each species.</p></div

Distribution of functional categories in <i>T. pedis</i> strain T A4 and <i>T. denticola</i> strain ATCC 35405 according to the COG classification.

<p>Distribution of functional categories in <i>T. pedis</i> strain T A4 and <i>T. denticola</i> strain ATCC 35405 according to the COG classification.</p

Proteases identified in <i>T. pedis</i> T A4 and their putative catalytic residues.

<p>Proteases identified in <i>T. pedis</i> T A4 and their putative catalytic residues.</p

Quantification of strain-specific, intermediately-represented, and core genes in <i>T. pedis</i> and <i>T. denticola</i>.

<p>The distribution between strain-specific genes, intermediately-represented genes, and genes present in all genomes, i.e., core functions, in the analyzed genome datasets of <i>T. pedis</i> (A) and <i>T. denticola</i> (B). The gene representation was determined by a clustering analysis that collapsed CDSs sharing >80% BLASTP identity and that had <30% length difference.</p

Circular representation of the <i>T. pedis</i> T A4 genome and complete genome alignment with <i>T. denticola</i>.

<p>(A.) Circular representation of the <i>T. pedis</i> T A4 genome. The CDSs are shown in violet where the outer circle represents predictions on the plus strand and the second circle those on the minus strand. CDSs with a best BLASTP hit in <i>T. denticola</i> ATCC 35405 are colored red and shown in the third circle. The fourth circle represents genes with best BLASTP hits in <i>T. brennaborense</i> (black), <i>F. nucleatum</i> (green), <i>F. alocis</i> (blue) and <i>T. succinifaciens</i> (grey). G+C skew is drawn in the inner circle. (B.) Complete genome alignment between <i>T. pedis</i> T A4 and <i>T. denticola</i> ATCC 35405. Dots represent maximum unique matches (MUMs) between the genomes. MUMs oriented in the same direction are depicted as red dots and reverse complemented MUMs are depicted as blue dots.</p

Phylogeny of <i>T. pedis</i> isolates and <i>T. denticola</i> strains.

<p>Phylogeny based on neighbor-joining of the intergenic spacer region between the 16S rRNA and tRNA^Ile. The sequences were extracted from the WGS assemblies generated in this study. (A) Phylogeny of the <i>T. pedis</i> isolates. (B) Phylogeny of the <i>T. denticola</i> strains. The bars corresponds to 0.015 and 0.050 nucleotide substitutions per position.</p

Ninety-nine de novo assembled genomes from the moose (Alces alces) rumen microbiome provide new insights into microbial plant biomass degradation

International audienceThe moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries