Borrelia burgdorferi infection induces long-term memory-like responses in macrophages with tissue-wide consequences in the heart.

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo.

Summary-data based Mendelian randomization identifies gene expression regulatory polymorphisms associated with bovine paratuberculosis by modulation of the nuclear factor Kappa ß (NF-κß)-mediated inflammatory response.

Genome-wide association studies (GWAS) have identified host genetic variants associated with paratuberculosis (PTB) susceptibility. Most of the GWAS-identified SNPs are in non-coding regions. Connecting these non-coding variants and downstream affected genes is a challenge and, up to date, only a few functional mutations or expression quantitative loci (cis-eQTLs) associated with PTB susceptibility have been identified. In the current study, the associations between imputed whole-genome sequence genotypes and whole RNA-Sequencing data from peripheral blood (PB) and ileocecal valve (ICV) samples of Spanish Holstein cows (N = 16) were analyzed with TensorQTL. This approach allowed the identification of 88 and 37 cis-eQTLs regulating the expression levels of 90 and 37 genes in PB and ICV samples, respectively (False discorey rate, FDR ≤ 0.05). Next, we applied summary-based data Mendelian randomization (SMR) to integrate the cis-eQTL dataset with GWAS data obtained from a cohort of 813 culled cattle that were classified according to the presence or absence of PTB-associated histopathological lesions in gut tissues. After multiple testing corrections (FDR ≤ 0.05), we identified two novel cis-eQTLs affecting the expression of the early growth response factor 4 (EGR4) and the bovine neuroblastoma breakpoint family member 6-like protein isoform 2 (MGC134040) that showed pleiotropic associations with the presence of multifocal and diffuse lesions in gut tissues; P = 0.002 and P = 0.017, respectively. While EGR4 acts as a brake on T-cell proliferation and cytokine production through interaction with the nuclear factor Kappa ß (NF-κß), MGC134040 is a target gene of NF-κß. Our findings provide a better understanding of the genetic factors influencing PTB outcomes, confirm that the multifocal lesions are localized/confined lesions that have different underlying host genetics than the diffuse lesions, and highlight regulatory SNPs and regulated-gene targets to design future functional studies.

Reduction of the resistome risk from cow slurry and manure microbiomes to soil and vegetable microbiomes.

In cow farms, the interaction between animal and environmental microbiomes creates hotspots for antibiotic resistance dissemination. A shotgun metagenomic approach was used to survey the resistome risk in five dairy cow farms. To this purpose, 10 environmental compartments were sampled: 3 of them linked to productive cows (fresh slurry, stored slurry, slurry-amended pasture soil); 6 of them to non-productive heifers and dry cows (faeces, fresh manure, aged manure, aged manure-amended orchard soil, vegetables-lettuces and grazed soil); and, finally, unamended control soil. The resistome risk was assessed using MetaCompare, a computational pipeline which scores the resistome risk according to possible links between antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and human pathogens. The resistome risk decreased from slurry and manure microbiomes to soil and vegetable microbiomes. In total (sum of all the compartments), 18,157 ARGs were detected: 24% related to ansamycins, 21% to multidrugs, 14% to aminoglycosides, 12% to tetracyclines, 9% to ß-lactams, and 9% to macrolide-lincosamide-streptogramin B. All but two of the MGE-associated ARGs were only found in the animal dejections (not in soil or vegetable samples). Several ARGs with potential as resistome risk markers (based on their presence in hubs of co-occurrence networks and high dissemination potential) were identified. As a precautionary principle, improved management of livestock dejections is necessary to minimize the risk of antibiotic resistance.

Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Glucocorticoids (GCs) are important hormones involved in the regulation of multiple physiologic functions. GCs are also widely used in anti-inflammatory/immunosuppressant drugs. GCs are synthesized by the adrenal cortex as part of the hypothalamus-pituitary-adrenal axis and also by intestinal epithelial cells, among other peripheral sites. GCs are one of the main therapy choices for the exacerbations of inflammatory bowel disease, but they are not useful to prolong remission, and development of tolerance with secondary treatment failure is frequent. Thus, GC actions at the intestinal epithelial level are of great importance, both physiologically and pharmacologically. We generated a tamoxifen-inducible nuclear receptor subfamily 3 group C member 1 (NR3C1)ΔIEC mouse model to study the effects of GCs on epithelial cells in vivo. Nr3c1 deletion in epithelial cells of the small intestine and colon was associated with limited colonic inflammation at 1 wk postdeletion, involving augmented epithelial proliferation and mucus production, plus local and systemic immune/inflammatory changes. This phenotype regressed substantially, but not completely, after 2 wk. The mechanism may involve augmented inflammatory signaling by epithelial cells or defective barrier function. We conclude that the epithelial GC receptor plays a significant role in colonic homeostasis in basal conditions, but its deficiency can be compensated in the short term. Future studies are required to assess the impact of Nr3c1 deletion in other conditions such as experimental colitis.-Aranda, C. J., Arredondo-Amador, M., Ocón, B., Lavín, J. L., Aransay, A. M., Martínez-Augustin, O., Sánchez de Medina, F. Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Cluster Locator, online analysis and visualization of gene clustering.

Summary: Genes sharing functions, expression patterns or quantitative traits are not randomly distributed along eukaryotic genomes. In order to study the distribution of genes that share a given feature, we present Cluster Locator, an online analysis and visualization tool. Cluster Locator determines the number, size and position of all the clusters formed by the protein-coding genes on a list according to a given maximum gap, the percentage of gene clustering of the list and its statistical significance. The output includes a visual representation of the distribution of genes and gene clusters along the reference genome. Availability and implementation: Cluster Locator is freely available at http://clusterlocator.bnd.edu.uy/. Supplementary information: Supplementary data are available at Bioinformatics online.

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum.

BACKGROUND: Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds. RESULTS: We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanBFnp). TanBFnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanBFnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanBFnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanBFnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanBFnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications. CONCLUSIONS: We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanBFnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.

Whole-genome long-read sequencing to unveil Enterococcus antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of Enterococcus lactis.

Enterococcus faecalis (Efs) and Enterococcus faecium (Efm) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis (Elc). However, identification techniques do not routinely differentiate Elc from Efm. As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs, 23 Efm, and 59 Elc. Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCEEnterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis (Elc) from Enterococcus faecium (Efm) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis (Efs) and Efm, it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (Efs, Efm, and Elc) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

Corrigendum: Alternative splicing of pre-mRNA modulates the immune response in Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

[This corrects the article DOI: 10.3389/fimmu.2024.1354500.].

Alternative splicing of pre-mRNA modulates the immune response in Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

Little is known about the role of alternative splicing (AS) in regulating gene expression in Mycobacteria-infected individuals in distinct stages of infection. Pre-mRNA AS consists of the removal of introns and the assembly of exons contained in eukaryotic genes. AS events can influence transcript stability or structure with important physiological consequences. Using RNA-Seq data from peripheral blood (PB) and ileocecal valve (ICV) samples collected from Holstein cattle with focal and diffuse paratuberculosis (PTB)-associated histopathological lesions in gut tissues and without lesions (controls), we detected differential AS profiles between the infected and control groups. Four of the identified AS events were experimentally validated by reverse transcription-digital droplet PCR (RT-ddPCR). AS events in several genes correlated with changes in gene expression. In the ICV of animals with diffuse lesions, for instance, alternatively spliced genes correlated with changes in the expression of genes involved in endocytosis, antigen processing and presentation, complement activation, and several inflammatory and autoimmune diseases in humans. Taken together, our results identified common mechanisms of AS involvement in the pathogenesis of PTB and human diseases and shed light on novel diagnostic and therapeutic interventions to control these diseases.

Comparative pangenomic analysis of Campylobacter fetus isolated from Spanish bulls and other mammalian species.

Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). The latter causes bovine genital campylobacteriosis, a sexually-transmitted disease endemic in Spain that results in significant economic losses in the cattle industry. Here, 33 C. fetus Spanish isolates were whole-genome sequenced and compared with 62 publicly available C. fetus genomes from other countries. Genome-based taxonomic identification revealed high concordance with in silico PCR, confirming Spanish isolates as Cff (n = 4), Cfv (n = 9) and Cfv biovar intermedius (Cfvi, n = 20). MLST analysis assigned the Spanish isolates to 6 STs, including three novel: ST-76 and ST-77 for Cfv and ST-78 for Cff. Core genome SNP phylogenetic analysis of the 95 genomes identified multiple clusters, revealing associations at subspecies and biovar level between genomes with the same ST and separating the Cfvi genomes from Spain and other countries. A genome-wide association study identified pqqL as a Cfv-specific gene and a potential candidate for more accurate identification methods. Functionality analysis revealed variations in the accessory genome of C. fetus subspecies and biovars that deserve further studies. These results provide valuable information about the regional variants of C. fetus present in Spain and the genetic diversity and predicted functionality of the different subspecies.

Monitoring within-farm transmission dynamics of antimicrobial-resistant Campylobacter in dairy cattle using broth microdilution and long-read whole genome sequencing.

Campylobacter jejuni and Campylobacter coli are important foodborne zoonotic pathogens and cause for concern due to the increasing trend in antimicrobial resistance. A long-run surveillance study was conducted in animals from different age groups in five dairy cattle farms to investigate the within-farm diversity and transmission dynamics of resistant Campylobacter throughout time. The resistance phenotype of the circulating isolates (170 C. jejuni and 37 C. coli) was determined by broth microdilution and a selection of 56 isolates were whole genome sequenced using the Oxford-Nanopore long-fragment sequencing technology resulting in completely resolved and circularized genomes (both chromosomes and plasmids). C. jejuni was isolated from all farms while C. coli was isolated from only two farms, but resistance rates were higher in C. coli than in C. jejuni and in calves than in adult animals. Some genotypes (e.g. ST-48, gyrA_T86I/tet(O)/blaOXA-61 in farm F1; ST-12000, aadE-Cc/tet(O)/blaOXA-489 in F4) persisted throughout the study while others were only sporadically detected. Acquisition of extracellular genes from other isolates and intracellular mutational events were identified as the processes that led to the emergence of the resistant genotypes that spread within the herds. Monitoring with Oxford Nanopore Technologies sequencing helped to decipher the complex molecular epidemiology underlying the within-farm dissemination of resistant Campylobacter.

The Ixodes ricinus salivary gland proteome during feeding and B. Afzelii infection: New avenues for an anti-tick vaccine.

INTRODUCTION: Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. METHOD: Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. RESULTS: We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. CONCLUSION: Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine.

Mitochondrial dysfunction promotes microbial composition that negatively impacts on ulcerative colitis development and progression.

Recent evidence demonstrates potential links between mitochondrial dysfunction and inflammatory bowel diseases (IBD). In addition, bidirectional interactions between the intestinal microbiota and host mitochondria may modulate intestinal inflammation. We observed previously that mice deficient in the mitochondrial protein MCJ (Methylation-controlled J protein) exhibit increased susceptibility to DSS colitis. However, it is unclear whether this phenotype is primarily driven by MCJ-/- associated gut microbiota dysbiosis or by direct effects of MCJ-deficiency. Here, we demonstrate that fecal microbiota transplantation (FMT) from MCJ-deficient into germ-free mice was sufficient to confer increased susceptibility to colitis. Therefore, an FMT experiment by cohousing was designed to alter MCJ-deficient microbiota. The phenotype resulting from complex I deficiency was reverted by FMT. In addition, we determined the protein expression pathways impacted by MCJ deficiency, providing insight into the pathophysiology of IBD. Further, we used magnetic activated cell sorting (MACS) and 16S rRNA gene sequencing to characterize taxa-specific coating of the intestinal microbiota with Immunoglobulin A (IgA-SEQ) in MCJ-deficient mice. We show that high IgA coating of fecal bacteria observed in MCJ-deficient mice play a potential role in disease progression. This study allowed us to identify potential microbial signatures in feces associated with complex I deficiency and disease progression. This research highlights the importance of finding microbial biomarkers, which might serve as predictors, permitting the stratification of ulcerative colitis (UC) patients into distinct clinical entities of the UC spectrum.

Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis.

Anti-TNF therapy can induce and maintain a remission status during intestinal bowel disease. However, up to 30% of patients do not respond to this therapy by mechanisms that are unknown. Here, we show that the absence of MCJ, a natural inhibitor of the respiratory chain Complex I, induces gut microbiota changes that are critical determinants of the lack of response in a murine model of DSS-induced inflammation. First, we found that MCJ expression is restricted to macrophages in human colonic tissue. Therefore, we demonstrate by transcriptomic analysis of colon macrophages from DSS-induced mice that MCJ-deficiency is linked to the expression of genes belonging to the FcγR signaling pathway and contains an anti-TNF refractory gene signature identified in ulcerative colitis patients. The gut microbial composition changes observed upon DSS treatment in the MCJ-deficient mice revealed the increased presence of specific colitogenic members, including Ruminococcus gnavus and Oscillospira, which could be associated with the non-response to TNF inhibitors. Further, we show that the presence of a microbiota associated resistance to treatment is dominant and transmissible to responsive individuals. Collectively, our findings underscore the critical role played by macrophage mitochondrial function in the gut ecological niche that can substantially affect not only the severity of inflammation but also the ability to successfully respond to current therapies.

Within-farm dynamics of ESBL-producing Escherichia coli in dairy cattle: Resistance profiles and molecular characterization by long-read whole-genome sequencing.

A longitudinal study was designed in five dairy cattle farms to assess the within-farm dynamics of ESBL-/AmpC-/carbapenemase-producing E. coli and their resistance profiles, along with the genes conferring the resistance phenotypes. Twelve samplings were performed over a period of 16 months, collecting rectal feces from apparently healthy animals in three age groups (calves, heifers, and lactating cows) that were subjected to selective isolation in cefotaxime-containing media. Minimum inhibitory concentrations were determined by broth microdilution for 197 cefotaxime-resistant E. coli (1-3 isolates per age group and sampling date), and 41 of them were selected for long-read whole-genome sequencing. Cefotaxime-resistant E. coli were detected in the five farms, but isolation frequency and resistance profiles varied among farms and age groups. The genetic profiling of a selection of isolates recovered in two of the farms was described in full detail, showing the predominance of a few genomic subtypes of E. coli in one farm (F1) and great variability of strains in another one (F4). Two predominant distinct strains carrying the bla CTX-M-1 gene in IncX1 plasmids successively spread and persisted in F1 over a prolonged period. In F4, 13 different MLST types carrying a high diversity of ESBL-encoding genes in 6 different plasmid types were observed, probably as the result of multiple source contamination events. In both farms, the presence of certain plasmid types with the same repertoire of ARGs in different E. coli STs strongly suggested the occurrence of horizontal transfer of such plasmids among strains circulating within the farms. Considering the public health importance of ESBL-producing E. coli both as pathogens and as vectors for resistance mechanisms, the presence of ß-lactamase- and other AMR-encoding genes in plasmids that can be readily transferred between bacteria is a concern that highlights the need for One Health surveillance.

Mitochondrial complex I dysfunction alters the balance of soluble and membrane-bound TNF during chronic experimental colitis.

Inflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor α converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function.

Genomics and transcriptomics characterization of genes expressed during postharvest at 4°C by the edible basidiomycete Pleurotus ostreatus.

Pleurotus ostreatus is an industrially cultivated basidiomycete with nutritional and environmental applications. Its genome, which was sequenced by the Joint Genome Institute, has become a model for lignin degradation and for fungal genomics and transcriptomics studies. The complete P. ostreatus genome contains 35 Mbp organized in 11 chromosomes, and two different haploid genomes have been individually sequenced. In this work, genomics and transcriptomics approaches were employed in the study of P. ostreatus under different physiological conditions. Specifically, we analyzed a collection of expressed sequence tags (EST) obtained from cut fruit bodies that had been stored at 4°C for 7 days (postharvest conditions). Studies of the 253 expressed clones that had been automatically and manually annotated provided a detailed picture of the life characteristics of the self-sustained fruit bodies. The results suggested a complex metabolism in which autophagy, RNA metabolism, and protein and carbohydrate turnover are increased. Genes involved in environment sensing and morphogenesis were expressed under these conditions. The data improve our understanding of the decay process in postharvest mushrooms and highlight the use of high-throughput techniques to construct models of living organisms subjected to different environmental conditions.

Whole genome-based characterisation of antimicrobial resistance and genetic diversity in Campylobacter jejuni and Campylobacter coli from ruminants.

Campylobacter, a leading cause of gastroenteritis in humans, asymptomatically colonises the intestinal tract of a wide range of animals.Although antimicrobial treatment is restricted to severe cases, the increase of antimicrobial resistance (AMR) is a concern. Considering the significant contribution of ruminants as reservoirs of resistant Campylobacter, Illumina whole-genome sequencing was used to characterise the mechanisms of AMR in Campylobacter jejuni and Campylobacter coli recovered from beef cattle, dairy cattle, and sheep in northern Spain. Genome analysis showed extensive genetic diversity that clearly separated both species. Resistance genotypes were identified by screening assembled sequences with BLASTn and ABRicate, and additional sequence alignments were performed to search for frameshift mutations and gene modifications. A high correlation was observed between phenotypic resistance to a given antimicrobial and the presence of the corresponding known resistance genes. Detailed sequence analysis allowed us to detect the recently described mosaic tet(O/M/O) gene in one C. coli, describe possible new alleles of blaOXA-61-like genes, and decipher the genetic context of aminoglycoside resistance genes, as well as the plasmid/chromosomal location of the different AMR genes and their implication for resistance spread. Updated resistance gene databases and detailed analysis of the matched open reading frames are needed to avoid errors when using WGS-based analysis pipelines for AMR detection in the absence of phenotypic data.