Genomic and Immunological Characterization of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Isolates from Northwest Argentina.

In recent years, an increase in the prevalence hypermucoviscous carbapenem-resistant Klebsiella pneumoniae with sequence type 25 (ST25) was detected in hospitals of Tucuman (Northwest Argentina). In this work, the virulence and the innate immune response to two K. pneumoniae ST25 strains (LABACER 01 and LABACER 27) were evaluated in a murine model after a respiratory challenge. In addition, comparative genomics was performed with K. pneumoniae LABACER01 and LABACER27 to analyze genes associated with virulence. Both LABACER01 and LABACER27 were detected in the lungs of infected mice two days after the nasal challenge, with LABACER01 counts significantly higher than those of LABACER27. Only LABACER01 was detected in hemocultures. Lactate dehydrogenase (LDH) and albumin levels in bronchoalveolar lavage (BAL) samples were significantly higher in mice challenged with LABACER01 than in LABACER27-infected animals, indicating greater lung tissue damage. Both strains increased the levels of neutrophils, macrophages, TNF-α, IL-1ß, IL-6, KC, MCP-1, IFN-γ, and IL-17 in the respiratory tract and blood, with the effect of LABACER01 more marked than that of LABACER27. In contrast, LABACER27 induced higher levels of IL-10 in the respiratory tract than LABACER01. Genomic analysis revealed that K. pneumoniae LABACER01 and LABACER27 possess virulence factors found in other strains that have been shown to be hypervirulent, including genes required for enterobactin (entABCDEF) and salmochelin (iroDE) biosynthesis. In both strains, the genes of toxin-antitoxin systems, as well as regulators of the expression of virulence factors and adhesion genes were also detected. Studies on the genetic potential of multiresistant K. pneumoniae strains as well as their cellular and molecular interactions with the host are of fundamental importance to assess the association of certain virulence factors with the intensity of the inflammatory response. In this sense, this work explored the virulence profile based on genomic and in vivo studies of hypermucoviscous carbapenem-resistant K. pneumoniae ST25 strains, expanding the knowledge of the biology of the emerging ST25 clone in Argentina.

Stool and Ruminal Microbiome Components Associated With Methane Emission and Feed Efficiency in Nelore Beef Cattle.

Background: The impact of extreme changes in weather patterns on the economy and human welfare is one of the biggest challenges our civilization faces. From anthropogenic contributions to climate change, reducing the impact of farming activities is a priority since it is responsible for up to 18% of global greenhouse gas emissions. To this end, we tested whether ruminal and stool microbiome components could be used as biomarkers for methane emission and feed efficiency in bovine by studying 52 Brazilian Nelore bulls belonging to two feed intervention treatment groups, that is, conventional and by-product-based diets. Results: We identified a total of 5,693 amplicon sequence variants (ASVs) in the Nelore bulls' microbiomes. A Differential abundance analysis with the ANCOM approach identified 30 bacterial and 15 archaeal ASVs as differentially abundant (DA) among treatment groups. An association analysis using Maaslin2 software and a linear mixed model indicated that bacterial ASVs are linked to the host's residual methane emission (RCH4) and residual feed intake (RFI) phenotype variation, suggesting their potential as targets for interventions or biomarkers. Conclusion: The feed composition induced significant differences in both abundance and richness of ruminal and stool microbial populations in ruminants of the Nelore breed. The industrial by-product-based dietary treatment applied to our experimental groups influenced the microbiome diversity of bacteria and archaea but not of protozoa. ASVs were associated with RCH4 emission and RFI in ruminal and stool microbiomes. While ruminal ASVs were expected to influence CH4 emission and RFI, the relationship of stool taxa, such as Alistipes and Rikenellaceae (gut group RC9), with these traits was not reported before and might be associated with host health due to their link to anti-inflammatory compounds. Overall, the ASVs associated here have the potential to be used as biomarkers for these complex phenotypes.

The structure of microbial populations in Nelore GIT reveals inter-dependency of methanogens in feces and rumen.

BACKGROUND: The success of different species of ruminants in the colonization of a diverse range of environments is due to their ability to digest and absorb nutrients from cellulose, a complex polysaccharide found in leaves and grass. Ruminants rely on a complex and diverse microbial community, or microbiota, in a unique compartment known as the rumen to break down this polysaccharide. Changes in microbial populations of the rumen can affect the host's development, health, and productivity. However, accessing the rumen is stressful for the animal. Therefore, the development and use of alternative sampling methods are needed if this technique is to be routinely used in cattle breeding. To this end, we tested if the fecal microbiome could be used as a proxy for the rumen microbiome due to its accessibility. We investigated the taxonomic composition, diversity and inter-relations of two different GIT compartments, rumen and feces, of 26 Nelore (Bos indicus) bulls, using Next Generation Sequencing (NGS) metabarcoding of bacteria, archaea and ciliate protozoa. RESULTS: We identified 4265 Amplicon Sequence Variants (ASVs) from bacteria, 571 from archaea, and 107 from protozoa, of which 143 (96 bacteria and 47 archaea) were found common between both microbiomes. The most prominent bacterial phyla identified were Bacteroidetes (41.48%) and Firmicutes (56.86%) in the ruminal and fecal microbiomes, respectively, with Prevotella and Ruminococcaceae UCG-005 the most relatively abundant genera identified in each microbiome. The most abundant archaeal phylum identified was Euryarchaeota, of which Methanobrevibacter gottschalkii, a methanogen, was the prevalent archaeal species identified in both microbiomes. Protozoa were found exclusively identified in the rumen with Bozasella/Triplumaria being the most frequent genus identified. Co-occurrence among ruminal and fecal ASVs reinforces the relationship of microorganisms within a biological niche. Furthermore, the co-occurrence of shared archaeal ASVs between microbiomes indicates a dependency of the predominant fecal methanogen population on the rumen population. CONCLUSIONS: Co-occurring microorganisms were identified within the rumen and fecal microbiomes, which revealed a strong association and inter-dependency between bacterial, archaeal and protozoan populations of the same microbiome. The archaeal ASVs identified as co-occurring between GIT compartments corresponded to the methanogenic genera Methanobrevibacter and Methanosphaera and represented 26.34% of the overall archaeal sequencesdiversity in the rumen and 42.73% in feces. Considering that these archaeal ASVs corresponded to a significant part of the overall diversity of both microbiomes, which is much higher if one includes the interactions of these co-occurring with other rumen archaea ASVs, we suggest that fecal methanogens could be used as a proxy of ruminal methanogens.

Co-Expression Networks Reveal Potential Regulatory Roles of miRNAs in Fatty Acid Composition of Nelore Cattle.

Fatty acid (FA) content affects the sensorial and nutritional value of meat and plays a significant role in biological processes such as adipogenesis and immune response. It is well known that, in beef, the main FAs associated with these biological processes are oleic acid (C18:1 cis9, OA) and conjugated linoleic acid (CLA-c9t11), which may have beneficial effects on metabolic diseases such as type 2 diabetes and obesity. Here, we performed differential expression and co-expression analyses, weighted gene co-expression network analysis (WGCNA) and partial correlation with information theory (PCIT), to uncover the complex interactions between miRNAs and mRNAs expressed in skeletal muscle associated with FA content. miRNA and mRNA expression data were obtained from skeletal muscle of Nelore cattle that had extreme genomic breeding values for OA and CLA. Insulin and MAPK signaling pathways were identified by WGCNA as central pathways associated with both of these fatty acids. Co-expression network analysis identified bta-miR-33a/b, bta-miR-100, bta-miR-204, bta-miR-365-5p, bta-miR-660, bta-miR-411a, bta-miR-136, bta-miR-30-5p, bta-miR-146b, bta-let-7a-5p, bta-let-7f, bta-let-7, bta-miR 339, bta-miR-10b, bta-miR 486, and the genes ACTA1 and ALDOA as potential regulators of fatty acid synthesis. This study provides evidence and insights into the molecular mechanisms and potential target genes involved in fatty acid content differences in Nelore beef cattle, revealing new candidate pathways of phenotype modulation that could positively benefit beef production and human consumption.

The Resistome of Low-Impacted Marine Environments Is Composed by Distant Metallo-ß-Lactamases Homologs.

The worldwide dispersion and sudden emergence of new antibiotic resistance genes (ARGs) determined the need in uncovering which environment participate most as their source and reservoir. ARGs closely related to those currently found in human pathogens occur in the resistome of anthropogenic impacted environments. However, the role of pristine environment as the origin and source of ARGs remains underexplored and controversy, particularly, the marine environments represented by the oceans. Here, due to the ocean nature, we hypothesized that the resistome of this pristine/low-impacted marine environment is represented by distant ARG homologs. To test this hypothesis we performed an in silico analysis on the Global Ocean Sampling (GOS) metagenomic project dataset focusing on the metallo-ß-lactamases (MßLs) as the ARG model. MßLs have been a challenge to public health, since they hydrolyze the carbapenems, one of the last therapeutic choice in clinics. Using Hidden Markov Model (HMM) profiles, we were successful in identifying a high diversity of distant MßL homologs, related to the B1, B2, and B3 subclasses. The majority of them were distributed across the Atlantic, Indian, and Pacific Oceans being related to the chromosomally encoded MßL GOB present in Elizabethkingia genus. It was observed only a reduced number of metagenomic sequence homologs related to the acquired MßL enzymes (VIM, SPM-1, and AIM-1) that currently have impact in clinics. Therefore, low antibiotic impacted marine environment, as the ocean, are unlikely the source of ARGs that have been causing enormous threat to the public health.

Polyketide synthase gene diversity within the microbiome of the sponge Arenosclera brasiliensis, endemic to the Southern Atlantic Ocean.

Microbes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida sponge Arenosclera brasiliensis hosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome of A. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel and A. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes in A. brasiliensis as well as the potential of its microbiome to serve as a source of new polyketides.

Algorithms to predict cerebral malaria in murine models using the SHIRPA protocol.

BACKGROUND: Plasmodium berghei ANKA infection in C57Bl/6 mice induces cerebral malaria (CM), which reproduces, to a large extent, the pathological features of human CM. However, experimental CM incidence is variable (50-100%) and the period of incidence may present a range as wide as 6-12 days post-infection. The poor predictability of which and when infected mice will develop CM can make it difficult to determine the causal relationship of early pathological changes and outcome. With the purpose of contributing to solving these problems, algorithms for CM prediction were built. METHODS: Seventy-eight P. berghei-infected mice were daily evaluated using the primary SHIRPA protocol. Mice were classified as CM+ or CM- according to development of neurological signs on days 6-12 post-infection. Logistic regression was used to build predictive models for CM based on the results of SHIRPA tests and parasitaemia. RESULTS: The overall CM incidence was 54% occurring on days 6-10. Some algorithms had a very good performance in predicting CM, with the area under the receiver operator characteristic ((au)ROC) curve > or = 80% and positive predictive values (PV+) > or = 95, and correctly predicted time of death due to CM between 24 and 72 hours before development of the neurological syndrome ((au)ROC = 77-93%; PV+ = 100% using high cut off values). Inclusion of parasitaemia data slightly improved algorithm performance. CONCLUSION: These algorithms work with data from a simple, inexpensive, reproducible and fast protocol. Most importantly, they can predict CM development very early, estimate time of death, and might be a valuable tool for research using CM murine models.