Genomic characterization of New Delhi metallo-beta-lactamase-producing species of Morganellaceae, Yersiniaceae, and Enterobacteriaceae (other than Klebsiella) from Brazil over 2013-2022.

Over the last decade, New Delhi metallo-beta-lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed-field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM-1 allele was predominant (n = 29), but blaNDM-5 was identified in an E. coli specimen with a novel ST, and the blaNDM-7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr-1-producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended-spectrum ß-lactamases were common: cefotaximase from Munich (CTX-M)-15 (n = 10), CTX-M-2 (n = 4), and CTX-M-8 (n = 3) were detected, and the mcr-1-producing E. coli was found to co-produce both CTX-M-8 and CTX-M-55 ß-lactamases. The mcr-9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM-producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options.

Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples.

Streptococcus pneumoniae causes invasive diseases of significant public health concern, such as meningitis. The culture of cerebrospinal fluid (CSF) samples, the standard technique for meningitis diagnoses, is not always positive. Consequently, meaningful information about the etiological agent is lost, which can compromise effective epidemiological surveillance and the improvement of immunization policies. This study aims to standardize a method to genotype pneumococcus in the CSF samples which could mitigate the absence of isolated strains, and also evaluate the prediction of this assay. We applied eight multiplex PCR (mPCR) assays to CSF samples paired with the Quellung reaction applied to the isolated strains. We also compared different master mix kits in the mPCR. Moreover, a retrospective study was conducted with CSF samples considered pneumococcus positive due to the presence of the lytA gene. Results showed that genotyping by the mPCR correlated 100% with the Quellung reaction, and genotyping was dependent on the master mix applied. In the retrospective study (2014-2020), 73.4% were successfully genotyped. The analyses of the receiver operating characteristic curve showed that the cycle threshold (Ct value) around 30 for the lytA gene had a 75% positive chance of successful genotyping, whereas with a Ct value > 35, the chance was 12.5%. Finally, we observed that genotype 19A was prevalent in the period (12%), information unknown until now due to the lack of isolated strains. Therefore, the mPCR of CSF samples can efficiently predict S. pneumoniae serotypes, especially in the absence of isolated strains, which can be a great tool for pneumococcal serotype surveillance.

In silico and in vitro structure-stability-function relationship of analog peptides of Stigmurin and its antibacterial and antibiofilm activities.

Multidrug-resistant bacterial infections are a threat to public health worldwide, which boosts the urgent need for pharmacological research for new drugs. Although the peptides without disulfide bridges from scorpions have shown antimicrobial action, usually their toxicity hamper their pharmacological application. Stigmurin is a non-hemolytic cationic peptide from Tityus stigmurus venom with antibacterial effect and toxicity on normal cells. In this approach, the conformational changes and stability of two Stigmurin analog peptides, named StigA8 and StigA18, were evaluated by circular dichroism, as well as the mechanism of interaction with bacterial membranes in silico. In addition, the in vitro and in vivo antibacterial activity and the action against the biofilm formed by multidrug-resistant Staphylococcus aureus were investigated. StigA8 (+4) and StigA18 (+5) revealed the ability to change their structural conformation depending on the medium composition, and high stability at different temperatures and pH conditions. Both analog peptides showed greater ability to interact with bacterial membranes in silico when compared to the native one. StigA8 and StigA18 demonstrated low hemolytic action, with non-toxic effect on G. mellonella larvae up to 120 mg/kg. StigA8 and StigA18 presented a broad spectrum of antibacterial action in vitro, especially against multidrug-resistant clinical isolates. The analog peptides (7.5 µM) also reduced the biofilm biomass of multidrug-resistant S. aureus, as well as increased the larval survival of the Galleria mellonella infected larvae. Therefore, StigA8 and StigA18 showed a beneficial potential in the treatment of bacterial infections, constituting promising bioactive components for the development of new antimicrobial agents.

Development of a pneumococcal conjugate vaccine based on chemical conjugation of polysaccharide serotype 6B to PspA.

The use of conjugate vaccines remains an effective intervention to prevent pneumococcal diseases. In order to expand vaccine coverage, the inclusion of pneumococcal proteins as carriers is a propitious alternative that has been explored over the past few years. In this study, pneumococcal surface protein A (PspA) clade 1, family 1 (PspA1) and clade 3, family 2 (PspA3) were used as carrier proteins for pneumococcal capsular polysaccharide serotype 6B (Ps6B). Employing an improved reductive amination chemistry, 50% of Ps6B was incorporated to each protein, PspA1 and PspA3. The effect of chemical modifications in Ps6B and PspA was assessed by an antigenicity assay and circular dichroism, respectively. Fragmentation and oxidation decreased the antigenicity of Ps6B while conjugation improved antigenicity. In the same manner, introduction of adipic acid dihydrazide (ADH) reduced PspA secondary structure content, which was partially restored after conjugation. Immunization of Ps6B-PspA1 and Ps6B-PspA3 conjugates in mice induced specific IgG antibodies against the Ps6B and the protein; and anti-PspA antibodies had functional activity against two pneumococcal strains with different serotypes. These results suggest that chemical coupling between Ps6B and PspA did not affect antigenic epitopes and support the further development of PspA as a carrier protein in pneumococcal conjugate vaccines to provide broader protection.

Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.

Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain.

Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.

First isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil.

BACKGROUND: Infections caused by Shewanella spp. have been increasingly reported worldwide. The advances in genomic sciences have enabled better understanding about the taxonomy and epidemiology of this agent. However, the scarcity of DNA sequencing data is still an obstacle for understanding the genus and its association with infections in humans and animals. RESULTS: In this study, we report the first isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil using the boot sock method, as well as the resistance profile of this strain to antimicrobials. The isolate was first identified as Shewanella putrefaciens, but after whole-genome sequencing it showed greater similarity with Shewanella algae. The strain showed resistance to 46.7% of the antimicrobials tested, and 26 resistance genes were identified in the genome. CONCLUSIONS: This report supports research made with Shewanella spp. and gives a step forward for understanding its taxonomy and epidemiology. It also highlights the risk of emerging pathogens with high resistance to antimicrobial formulas that are important to public health.

Chemokine expression profiles in liver and kidney of mice with different susceptibilities to leptospirosis.

Leptospirosis is a global disease that affects humans and animals, impacting public health and the economy. The symptoms caused by Leptospira infection can vary from mild to severe, affecting liver, lungs, and kidneys. The host-pathogen interaction in leptospirosis is still poorly understood, but there is evidence for the role of the host immune response in the pathogenesis. Chemokines are a family of structurally-related low-molecular-mass proteins (8-14 kDa) that signal the recruitment of leukocytes. In this study the profile of 22 chemokines were evaluated in liver and kidney of three mice strains with different phenotypes of susceptibility to leptospirosis. We extended our previously reported observations showing that expression of chemokines with homeostatic function, activation and chemotaxis of leukocytes are essential to modulate and to induce resistance to leptospirosis. Our findings support that an early induction of CXC chemokines in resistant BALB/c mice can be associated with the control of the infection. The correlation of chemokine expression between liver and kidney observed in BALB/c suggests that a balance of chemokine induction in the organs may contribute to resistance to leptospirosis.

Phagocytosis of Leptospira by leukocytes from mice with different susceptibility to leptospirosis and possible role of chemokines.

BACKGROUND: Leptospirosis is a widespread zoonosis caused by pathogenic prokaryotic microbes of the genus Leptospira. Although there are several reports in the literature, host-pathogen interaction is still poorly understood. The role of chemokine expression is important on the chemotaxis, activation and regulation of immune cells. Recent studies have shown that their expression profiles play an important role on the severity of leptospirosis outcome. We evaluated the phagocytosis of Leptospira by spleens cells from C3H/HeJ, C3H/HePas and BALB/c mouse strains, respectively susceptible, intermediate and resistant to leptospirosis, and by RAW 264.7 macrophages. Besides, we evaluated the effects of CCL2 treatment on the phagocytosis. The cells were incubated with or without CCL2 chemokine, and infected with virulent L. interrogans sv Copenhageni. Cells and culture supernatants were collected for subsequent analysis. RESULTS: The number of leptospires was higher in BALB/c cells, CCL2 pre-treated or only infected groups, when compared to C3H/HeJ and C3H/HePas cells. Indeed, CCL2 activation did not interfere in the phagocytosis of Leptospira. Expression of chemokines CXCL5 and CCL8 levels were significantly inhibited in infected BALB/c cells when compared to the non-infected control. CONCLUSIONS: Higher ability to phagocytosis and early modulation of some chemokines correlated with the resistance to leptospirosis disease. Exposure to CCL2 did not interfere on phagocytosis of Leptospira in our experimental conditions, but acted in the modulation of chemokines expression during Leptospira infection.

Potent and Broad-Spectrum Antimicrobial Activity of Analogs from the Scorpion Peptide Stigmurin.

Scorpion venom constitutes a rich source of biologically active compounds with high potential for therapeutic and biotechnological applications that can be used as prototypes for the design of new drugs. The aim of this study was to characterize the structural conformation, evaluate the antimicrobial activity, and gain insight into the possible action mechanism underlying it, for two new analog peptides of the scorpion peptide Stigmurin, named StigA25 and StigA31. The amino acid substitutions in the native sequence for lysine residues resulted in peptides with higher positive net charge and hydrophobicity, with an increase in the theoretical helical content. StigA25 and StigA31 showed the capacity to modify their structural conformation according to the environment, and were stable to pH and temperature variation-results similar to the native peptide. Both analog peptides demonstrated broad-spectrum antimicrobial activity in vitro, showing an effect superior to that of the native peptide, being non-hemolytic at the biologically active concentrations. Therefore, this study demonstrates the therapeutic potential of the analog peptides from Stigmurin and the promising approach of rational drug design based on scorpion venom peptide to obtain new anti-infective agents.

A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic.

Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244 µg/mL of rSm14, 181 µg/mL and 392 µg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147 µg/mL of rSm14 and 162 µg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.

Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.

Serotype-independent pneumococcal vaccines.

Streptococcus pneumoniae remains an important cause of disease with high mortality and morbidity, especially in children and in the elderly. The widespread use of the polysaccharide conjugate vaccines in some countries has led to a significant decrease in invasive disease caused by vaccine serotypes, but an increase in disease caused by non-vaccine serotypes has impacted on the overall efficacy of these vaccines on pneumococcal disease. The obvious solution to overcome such shortcomings would be the development of new formulations that provide serotype-independent immunity. This review focuses on the most promising approaches, including protein antigens, whole cell pneumococcal vaccines, and recombinant bacteria expressing pneumococcal antigens. The protective capacity of these vaccine candidates against the different stages of pneumococcal infection, including colonization, mucosal disease, and invasive disease in animal models is reviewed. Some of the human trials that have already been performed or that are currently ongoing are presented. Finally, the feasibility and the possible shortcomings of these candidates in relation to an ideal vaccine against pneumococcal infections are discussed.

Acquisition of plasmids from Shiga toxin-producing Escherichia coli strains had low or neutral fitness cost on commensal E. coli.

Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

Genomic Characterization of a Clinical NDM-1-Producing Klebsiella michiganensis from Brazil.

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.

Polymyxin Resistance in Salmonella: Exploring Mutations and Genetic Determinants of Non-Human Isolates.

Until 2015, polymyxin resistance was primarily attributed to chromosomal mutations. However, with the first report of mobile colistin resistance (mcr-1) in commensal Escherichia coli from food animals in China, the landscape has changed. To evaluate the presence of polymyxin resistance in Salmonella spp., a drop screening test for colistin and polymyxin B was carried out on 1156 isolates of non-human origin (animals, food, and the environment), received in Brazil, between 2016 and 2021. Subsequently, 210 isolates with resistant results in the drop test were subjected to the gold-standard test (broth microdilution) for both colistin and polymyxin B. Whole-genome sequencing (WGS) of 102 resistant isolates was performed for a comprehensive analysis of associated genes. Surprisingly, none of the isolates resistant to colistin in the drop test harbored any of the mcr variants (mcr-1 to mcr-10). WGS identified that the most common mutations were found in pmrA (n= 22; T89S) and pmrB (n = 24; M15T, G73S, V74I, I83A, A111V). Other resistance determinants were also detected, such as the aac(6')-Iaa gene in 72 isolates, while others carried beta-lactamase genes (blaTEM-1blaCTX-M-2, blaCMY-2). Additionally, genes associated with fluoroquinolone resistance (qnrB19, qnrS1, oqxA/B) were detected in 11 isolates. Colistin and polymyxin B resistance were identified among Salmonella from non-human sources, but not associated with the mcr genes. Furthermore, the already-described mutations associated with polymyxin resistance were detected in only a small number of isolates, underscoring the need to explore and characterize unknown genes that contribute to resistance.

Adhesin activity of Leptospira interrogans lipoprotein identified by in vivo and in vitro shotgun phage display.

Leptospira interrogans causes leptospirosis, one of the most common zoonotic diseases in the world. This pathogenic spirochete is able to bind to extracellular matrix, to express virulent factors and to cause host death. Until now, there is no effective human vaccine for the disease. Shotgun phage display genomic libraries of L. interrogans were constructed and used for in vivo biopanning in hamsters and screened for ligands able to bind to LLC-PK1 epithelial cells. In both panning procedures, clones coding for the putative lipoprotein LIC12976 were identified and, in order to confirm its adhesin activity, a recombinant protein was produced in Escherichia coli and showed to interact with A31 fibroblasts, LLC-PK1 and Vero epithelial cells in vitro. Moreover, rLIC12976 was shown to bind to laminin, indicating an adhesin function. This protein was also detected in extracts of L. interrogans from different serovars and it was found to be conserved among pathogenic leptospires. Further, the protein was tested as vaccine candidate and immunization of hamsters with LIC12976 did not confer protection against a lethal challenge with the homologous L. interrogans serovar Copenhageni. Nevertheless, LIC12976 seems to act as an adhesin, and may be important for the host-pathogen interaction, so that its study can contribute to the understanding of the virulence mechanisms in pathogenic leptospires.

Emergence of livestock-associated Mammaliicoccus sciuri ST71 co-harbouring mecA and mecC genes in Brazil.

The discovery and tracking of antimicrobial resistance genes are essential for understanding the evolution of bacterial resistance and restraining its dispersion. Mammaliicoccus sciuri (formerly Staphylococcus sciuri) is the most probable evolutionary repository of the mecA gene, that later disseminated to S. aureus. In this study, we describe the first double mecA/mecC homologue-positive non-aureus staphylococci and mammaliicocci (NASM) from the American continent, also representing the first report of mecC-positive NASM in Brazil. Two clonally related methicillin-resistant M. sciuri strains co-carrying mecA and mecC genes were isolated from the teat skin swab and milk sample collected from an ewe's left udder half. Both M. sciuri strains belonged to the sequence type (ST) 71. Besides mecA and mecC genes, the M. sciuri strains carried broad resistomes for clinically important antimicrobial agents, including ß-lactams, tetracyclines, lincosamide, streptogramin, streptomycin, and aminoglycosides. Virulome analysis showed the presence of the clumping factor B (clfB), ATP-dependent protease ClpP (ClpP) and serine-aspartate repeat proteins (sdrC and sdrE) virulence-associated genes. Phylogenomic analysis revealed that these M. sciuri strains are part of a globally disseminated branch, associated with farm and companion animals and even with food. Our findings suggest that M. sciuri is likely to emerge as a pathogen of global interest, carrying a broad repertoire of antimicrobial resistance genes with a remarkable co-presence of mecA and mecC genes. Finally, we strongly encourage to monitor M. sciuri under the One Health umbrella since this bacterial species is spreading at the human-animal-environment interface.