Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile.

BACKGROUND: The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. RESULTS: Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM-1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. CONCLUSIONS: We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the ß-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

Dominant Carbapenemase-Encoding Plasmids in Clinical Enterobacterales Isolates and Hypervirulent Klebsiella pneumoniae, Singapore.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010-2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

Exploiting the natural poly(3-hydroxyalkanoates) production capacity of Antarctic Pseudomonas strains: from unique phenotypes to novel biopolymers.

Extreme environments are a unique source of microorganisms encoding metabolic capacities that remain largely unexplored. In this work, we isolated two Antarctic bacterial strains able to produce poly(3-hydroxyalkanoates) (PHAs), which were classified after 16S rRNA analysis as Pseudomonas sp. MPC5 and MPC6. The MPC6 strain presented nearly the same specific growth rate whether subjected to a temperature of 4 °C 0.18 (1/h) or 30 °C 0.2 (1/h) on glycerol. Both Pseudomonas strains produced high levels of PHAs and exopolysaccharides from glycerol at 4 °C and 30 °C in batch cultures, an attribute that has not been previously described for bacteria of this genus. The MPC5 strain produced the distinctive medium-chain-length-PHA whereas Pseudomonas sp. MPC6 synthesized a novel polyoxoester composed of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate). Batch bioreactor production of PHAs in MPC6 resulted in a titer of 2.6 (g/L) and 1.3 (g/L), accumulating 47.3% and 34.5% of the cell dry mass as PHA, at 30 and 4 °C, respectively. This study paves the way for using Antarctic Pseudomonas strains for biosynthesizing novel PHAs from low-cost substrates such as glycerol and the possibility to carry out the bioconversion process for biopolymer synthesis without the need for temperature control.

Salmonella Typhimurium induces cloacitis-like symptomsin zebrafish larvae.

Pathogenic Salmonella strains have a set of virulence factors allowing them to generate systemic infections and damage in a variety of hosts. Among these factors, bacterial proteins secreted by specialized systems are used to penetrate the host's intestinal mucosa, through the invasion and destruction of specialized epithelial M cells in the intestine. On the other hand, numerous studies have demonstrated that humans, as well as experimental animal hosts, respond to Salmonella infection by activating both innate and adaptive immune responses. Here, through live cell imaging of S. Typhimurium infection of zebrafish larvae, we showed that besides the intestinal colonization, a deformed cloacae region and a concomitant accumulation of S. Typhimurium cells was observed upon bacterial infection. The swelling led to a persistent inflammation of infected larvae, although the infection was non-lethal. The in vivo inflammation process was confirmed by the co-localization of GFP-tagged S. Typhimurium with mCherry-tagged neutrophils at 72 h post exposition. Our live-cell analyses suggest that Salmonella Typhimurium induce cloacitis-like symptoms in zebrafish larvae.

Dictyostelium discoideum-assisted pharmacognosy of plant resources for discovering antivirulence molecules targeting Klebsiella pneumoniae.

The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to confront this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is a potent model organism in this effort. Employing this invertebrate model, we introduce a novel perspective to investigate natural plant extracts in search of molecules with potential antivirulence activity. Our work established an easy-scalable developmental assay targeting a virulent strain of Klebsiella pneumoniae, with Helenium aromaticum as the representative plant. The main objective was to identify tentative compounds from the Helenium aromaticum extract that attenuate the virulence of K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum fulfilled these prerequisites compared to the dichloromethane extract. Using UHPLC Q/Orbitrap/ESI/MS/MS, 63 compounds were tentatively identified in both extracts, 47 in the methanolic and 29 in the dichloromethane, with 13 compounds in common. This research underscores the potential of employing D. discoideum-assisted pharmacognosy to discover new antivirulence agents against multidrug-resistant pathogens.

Inorganic Polyphosphate Affects Biofilm Assembly, Capsule Formation, and Virulence of Hypervirulent ST23 Klebsiella pneumoniae.

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired infections in healthy individuals. The ability to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Polyphosphate kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a proteomic analysis to gain further insights into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins involved in capsule synthesis (Wzi-Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB-LpxC-PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hvKP. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

Hypervirulent Klebsiella pneumoniae employs genomic island encoded toxins against bacterial competitors in the gut.

The hypervirulent lineages of Klebsiella pneumoniae (HvKp) cause invasive infections such as Klebsiella-liver abscess. Invasive infection often occurs after initial colonization of the host gastrointestinal tract by HvKp. Over 80% of HvKp isolates belong to the clonal group 23 sublineage I that has acquired genomic islands (GIs) GIE492 and ICEKp10. Our analysis of 12 361 K. pneumoniae genomes revealed that GIs GIE492 and ICEKp10 are co-associated with the CG23-I and CG10118 HvKp lineages. GIE492 and ICEKp10 enable HvKp to make a functional bacteriocin microcin E492 (mccE492) and the genotoxin colibactin, respectively. We discovered that GIE492 and ICEKp10 play cooperative roles and enhance gastrointestinal colonization by HvKp. Colibactin is the primary driver of this effect, modifying gut microbiome diversity. Our in vitro assays demonstrate that colibactin and mccE492 kill or inhibit a range of Gram-negative Klebsiella species and Escherichia coli strains, including Gram-positive bacteria, sometimes cooperatively. Moreover, mccE492 and colibactin kill human anaerobic gut commensals that are similar to the taxa found altered by colibactin in the mouse intestines. Our findings suggest that GIs GIE492 and ICEKp10 enable HvKp to kill several commensal bacterial taxa during interspecies interactions in the gut. Thus, acquisition of GIE492 and ICEKp10 could enable better carriage in host populations and explain the dominance of the CG23-I HvKp lineage.

Microcin e492 amyloid formation is retarded by posttranslational modification.

Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.

Antimicrobial resistance, pathogenic potential, and genomic features of carbapenem-resistant Klebsiella pneumoniae isolated in Chile: high-risk ST25 clones and novel mobile elements.

Multidrug- and carbapenem-resistant Klebsiella pneumoniae (CR-Kp) are critical threats to global health and key traffickers of resistance genes to other pathogens. Despite the sustained increase in CR-Kp infections in Chile, few strains have been described at the genomic level, lacking details of their resistance and virulence determinants and the mobile elements mediating their dissemination. In this work, we studied the antimicrobial susceptibility and performed a comparative genomic analysis of 10 CR-Kp isolates from the Chilean surveillance of carbapenem-resistant Enterobacteriaceae. High resistance was observed among the isolates (five ST25, three ST11, one ST45, and one ST505), which harbored 44 plasmids, most carrying genes for conjugation and resistance to several antibiotics and biocides. Ten plasmids encoding carbapenemases were characterized, including novel plasmids or variants with additional resistance genes, a novel genetic environment for blaKPC-2, and plasmids widely disseminated in South America. ST25 K2 isolates belonging to CG10224, a clone traced back to 2012 in Chile, which recently acquired blaNDM-1, blaNDM-7, or blaKPC-2 plasmids stood out as high-risk clones. Moreover, this corresponds to the first report of ST25 and ST45 Kp producing NDM-7 in South America and ST505 CR-Kp producing both NDM-7 and KPC-2 worldwide. Also, we characterized a variety of genomic islands carrying virulence and fitness factors. These results provide baseline knowledge for a detailed understanding of molecular and genetic determinants behind antibiotic resistance and virulence of CR-Kp in Chile and South America. IMPORTANCE In the ongoing antimicrobial resistance crisis, carbapenem-resistant strains of Klebsiella pneumoniae are critical threats to public health. Besides globally disseminated clones, the burden of local problem clones remains substantial. Although genomic analysis is a powerful tool for improving pathogen and antimicrobial resistance surveillance, it is still restricted in low- to middle-income countries, including Chile, causing them to be underrepresented in genomic databases and epidemiology surveys. This study provided the first 10 complete genomes of the Chilean surveillance for carbapenem-resistant K. pneumoniae in healthcare settings, unveiling their resistance and virulence determinants and the mobile genetic elements mediating their dissemination, placed in the South American and global K. pneumoniae epidemiological context. We found ST25 with K2 capsule as an emerging high-risk clone, along with other lineages producing two carbapenemases and several other resistance and virulence genes encoded in novel plasmids and genomic islands.

Diversity, Taxonomic Novelty, and Encoded Functions of Salar de Ascotán Microbiota, as Revealed by Metagenome-Assembled Genomes.

Salar de Ascotán is a high-altitude arsenic-rich salt flat exposed to high ultraviolet radiation in the Atacama Desert, Chile. It hosts unique endemic flora and fauna and is an essential habitat for migratory birds, making it an important site for conservation and protection. However, there is limited information on the resident microbiota's diversity, genomic features, metabolic potential, and molecular mechanisms that enable it to thrive in this extreme environment. We used long- and short-read metagenomics to investigate the microbial communities in Ascotán's water, sediment, and soil. Bacteria predominated, mainly Pseudomonadota, Acidobacteriota, and Bacteroidota, with a remarkable diversity of archaea in the soil. Following hybrid assembly, we recovered high-quality bacterial (101) and archaeal (6) metagenome-assembled genomes (MAGs), including representatives of two putative novel families of Patescibacteria and Pseudomonadota and two novel orders from the archaeal classes Halobacteriota and Thermoplasmata. We found different metabolic capabilities across distinct lineages and a widespread presence of genes related to stress response, DNA repair, and resistance to arsenic and other metals. These results highlight the remarkable diversity and taxonomic novelty of the Salar de Ascotán microbiota and its rich functional repertoire, making it able to resist different harsh conditions. The highly complete MAGs described here could serve future studies and bioprospection efforts focused on salt flat extremophiles, and contribute to enriching databases with microbial genome data from underrepresented regions of our planet.

Assessment of Intracellular Amyloid Formation in Fixed and Live Bacteria Using Fluorescence Microscopy.

Although amyloid aggregation has been generally associated with protein misfolding and neurodegenerative diseases in mammals, bacteria and other organisms have harnessed amyloidogenesis to perform diverse biological processes. These functional amyloids, some of them secreted and others intracellular, require that the producing cells keep aggregation under control in the cytoplasm upon protein translation, preventing their inherent toxicity. Thus, it is highly relevant to understand how intracellular amyloid formation occurs and is regulated, its metabolic consequences, and the formation dynamics and fate of the amyloid inclusions upon cell division. This chapter describes methods leveraging fluorescence microscopy and fixed- or live-cell imaging to monitor intracellular amyloid formation in bacterial cells.

Identification of Aggregation-Prone and Gatekeeper Residues in Bacterial Amyloids Using Site-Directed Mutagenesis and Flow Cytometry.

Bacterial functional amyloids are remarkable examples of how amyloid aggregation can be kept under control and even leveraged to perform diverse biological processes. In this context, it is highly relevant to understand how amyloidogenesis is modulated by relevant factors, including key amino acids promoting or preventing aggregation. This chapter describes a methodology to identify critical residues for amyloid formation in bacterial proteins, based on mutant construction guided by bioinformatics prediction, their expression in bacteria, and their analysis by flow cytometry. Additionally, we describe a simple downstream analysis of selected mutants to assess their in vitro aggregation properties upon protein purification. We applied the proposed methodology to identify critical residues modulating the aggregation of the antimicrobial peptide microcin E492, a well-studied model of bacterial amyloids.

The highly diverse Antarctic Peninsula soil microbiota as a source of novel resistance genes.

The rise of multiresistant bacterial pathogens is currently one of the most critical threats to global health, encouraging a better understanding of the evolution and spread of antimicrobial resistance. In this regard, the role of the environment as a source of resistance mechanisms remains poorly understood. Moreover, we still know a minimal part of the microbial diversity and resistome present in remote and extreme environments, hosting microbes that evolved to resist harsh conditions and thus a potentially rich source of novel resistance genes. This work demonstrated that the Antarctic Peninsula soils host a remarkable microbial diversity and a widespread presence of autochthonous antibiotic-resistant bacteria and resistance genes. We observed resistance to a wide array of antibiotics among isolates, including Pseudomonas resisting ten or more different compounds, with an overall increased resistance in bacteria from non-intervened areas. In addition, genome analysis of selected isolates showed several genes encoding efflux pumps, as well as a lack of known resistance genes for some of the resisted antibiotics, including colistin, suggesting novel uncharacterized mechanisms. By combining metagenomic approaches based on analyzing raw reads, assembled contigs, and metagenome-assembled genomes, we found hundreds of widely distributed genes potentially conferring resistance to different antibiotics (including an outstanding variety of inactivation enzymes), metals, and biocides, hosted mainly by Polaromonas, Pseudomonas, Streptomyces, Variovorax, and Burkholderia. Furthermore, a proportion of these genes were found inside predicted plasmids and other mobile elements, including a putative OXA-like carbapenemase from Polaromonas harboring conserved key residues and predicted structural features. All this evidence indicates that the Antarctic Peninsula soil microbiota has a broad natural resistome, part of which could be transferred horizontally to pathogenic bacteria, acting as a potential source of novel resistance genes.

"Glucose and ethanol-dependent transcriptional regulation of the astaxanthin biosynthesis pathway in Xanthophyllomyces dendrorhous".

BACKGROUND: The yeast Xanthophyllomyces dendrorhous is one of the most promising and economically attractive natural sources of astaxanthin. The biosynthesis of this valuable carotenoid is a complex process for which the regulatory mechanisms remain mostly unknown. Several studies have shown a strong correlation between the carbon source present in the medium and the amount of pigments synthesized. Carotenoid production is especially low when high glucose concentrations are used in the medium, while a significant increase is observed with non-fermentable carbon sources. However, the molecular basis of this phenomenon has not been established. RESULTS: In this work, we showed that glucose caused transcriptional repression of the three genes involved in the synthesis of astaxanthin from geranylgeranyl pyrophosphate in X. dendrorhous, which correlates with a complete inhibition of pigment synthesis. Strikingly, this regulatory response was completely altered in mutant strains that are incapable of synthesizing astaxanthin. However, we found that addition of ethanol caused the induction of crtYB and crtS gene expression and promoted de novo synthesis of carotenoids. The induction of carotenogenesis was noticeable as early as 24 h after ethanol addition. CONCLUSION: For the first time, we demonstrated that carbon source-dependent regulation of astaxanthin biosynthesis in X. dendrorhous involves changes at the transcriptional level. Such regulatory mechanism provides an explanation for the strong and early inhibitory effect of glucose on the biosynthesis of this carotenoid.

Differential carotenoid production and gene expression in Xanthophyllomyces dendrorhous grown in a nonfermentable carbon source.

Xanthophyllomyces dendrorhous is a basidiomycetous yeast of considerable biotechnological interest because it synthesizes astaxanthin as its main carotenoid. The carotenoid production increases when it is grown using nonfermentable compounds as the sole carbon source. This work analyzes the expression of the carotenogenic genes and their relationship with the amount and types of carotenoids produced when X. dendrorhous is grown using a nonfermentable (succinate) or a fermentable carbon source (glucose). When X. dendrorhous is grown in succinate, carotenoid production is approximately three times higher than when it is grown in glucose. Moreover, carotenoid biosynthesis occurs at the start of the growth cycle when X. dendrorhous is grown in succinate, whereas when it is grown in glucose, carotenoids are produced at the end of the exponential phase. Additionally, we observed that some carotenogenic genes, such as alternative transcripts of crtYB and crtI, are differentially expressed when the yeast is grown in these carbon sources; other genes, such as crtS, exhibit a similar pattern of expression. Our data indicate that transcriptional regulation is not sufficient to explain the differences in carotenoid production between the two culture conditions, indicating that additional regulatory mechanisms may be operating in the carotenogenic pathway of X. dendrorhous.

Dysregulated Immune Responses in COVID-19 Patients Correlating With Disease Severity and Invasive Oxygen Requirements.

The prognosis of severe COVID-19 patients has motivated research communities to uncover mechanisms of SARS-CoV-2 pathogenesis also on a regional level. In this work, we aimed to understand the immunological dynamics of severe COVID-19 patients with different degrees of illness, and upon long-term recovery. We analyzed immune cellular subsets and SARS-CoV-2-specific antibody isotypes of 66 COVID-19 patients admitted to the Hospital Clínico Universidad de Chile, which were categorized according to the WHO ten-point clinical progression score. These included 29 moderate patients (score 4-5) and 37 severe patients under either high flow oxygen nasal cannula (18 patients, score 6), or invasive mechanical ventilation (19 patients, score 7-9), plus 28 convalescent patients and 28 healthy controls. Furthermore, six severe patients that recovered from the disease were longitudinally followed over 300 days. Our data indicate that severe COVID-19 patients display increased frequencies of plasmablasts, activated T cells and SARS-CoV-2-specific antibodies compared to moderate and convalescent patients. Remarkably, within the severe COVID-19 group, patients rapidly progressing into invasive mechanical ventilation show higher frequencies of plasmablasts, monocytes, eosinophils, Th1 cells and SARS-CoV-2-specific IgG than patients under high flow oxygen nasal cannula. These findings demonstrate that severe COVID-19 patients progressing into invasive mechanical ventilation show a distinctive type of immunity. In addition, patients that recover from severe COVID-19 begin to regain normal proportions of immune cells 100 days after hospital discharge and maintain high levels of SARS-CoV-2-specific IgG throughout the study, which is an indicative sign of immunological memory. Thus, this work can provide useful information to better understand the diverse outcomes of severe COVID-19 pathogenesis.

Exploiting Zebrafish Xenografts for Testing the in vivo Antitumorigenic Activity of Microcin E492 Against Human Colorectal Cancer Cells.

One of the approaches to address cancer treatment is to develop new drugs not only to obtain compounds with less side effects, but also to have a broader set of alternatives to tackle the resistant forms of this pathology. In this regard, growing evidence supports the use of bacteria-derived peptides such as bacteriocins, which have emerged as promising anti-cancer molecules. In addition to test the activity of these molecules on cancer cells in culture, their in vivo antitumorigenic properties must be validated in animal models. Although the standard approach for such assays employs experiments in nude mice, at the initial stages of testing, the use of high-throughput animal models would permit rapid proof-of-concept experiments, screening a high number of compounds, and thus increasing the possibilities of finding new anti-cancer molecules. A validated and promising alternative animal model are zebrafish larvae harboring xenografts of human cancer cells. Here, we addressed the anti-cancer properties of the antibacterial peptide microcin E492 (MccE492), a bacteriocin produced by Klebsiella pneumoniae, showing that this peptide has a marked cytotoxic effect on human colorectal cancer cells in vitro. Furthermore, we developed a zebrafish xenograft model using these cells to test the antitumor effect of MccE492 in vivo, demonstrating that intratumor injection of this peptide significantly reduced the tumor cell mass. Our results provide, for the first time, evidence of the in vivo antitumoral properties of a bacteriocin tested in an animal model. This evidence strongly supports the potential of this bacteriocin for the development of novel anti-cancer therapies.

Static Immersion and Injection Methods for Live Cell Imaging of Foodborne Pathogen Infections in Zebrafish Larvae.

Important features of host-pathogen interactions have been discovered using nonmammalian hosts. Therefore, model organisms such as the nematode Caenorhabditis elegans, the social amoeba Dictyostelium discoideum, and zebrafish ( Danio rerio ) have been increasingly used for studying bacterial pathogenesis in vivo. These host models are amenable for live cell imaging studies, which can also benefit from online resources and databases ( Dictybase.org , ZFIN.org , Wormbase.org ), as well as from a wide repertoire of genetic and genomic tools generated over the years by the scientific community. Here, we present the protocols we developed to study bacterial dynamics within infected embryonic zebrafish. This chapter describes detailed methods to achieve infections of zebrafish larvae with the foodborne pathogen Salmonella enterica serovar Typhimurium, including embryonic zebrafish spawning and maintenance, bacterial inoculation through intravenous injections and static immersion, followed by fluorescence imaging of infected transgenic zebrafish. Methods for studying bacterial dynamics within zebrafish larvae through live cell imaging are also described.

Inhibition of Escherichia coli and Bacillus subtilis FtsZ Polymerization and Bacillus subtilis Growth by Dihydroxynaphtyl Aryl Ketones.

The increasing detection of virulent and/or multidrug resistant bacterial strains makes necessary the development of new antimicrobial agents acting through novel mechanisms and cellular targets. A good choice are molecules aimed to interfere with the cell division machinery or divisome, which is indispensable for bacterial survival and propagation. A key component of this machinery, and thus a good target, is FtsZ, a highly conserved GTPase protein that polymerizes in the middle of the cell on the inner face of the cytoplasmic membrane forming the Z ring, which acts as a scaffold for the recruitment of the divisome proteins at the division site. In this work, we tested the inhibitory effect of five diaryl naphtyl ketone (dNAK) molecules on the in vitro polymerization of both Escherichia coli and Bacillus subtilis FtsZ (EcFtsZ and BsFtsZ, respectively). Among these compounds, dNAK 4 showed the strongest inhibition of FtsZ polymerization in vitro, with an IC50 of 2.3 ± 0.06 µM for EcFtsZ and 9.13 ± 0.66 µM for BsFtsZ. We found that dNAK 4 binds to GDP-FtsZ polymers but not to the monomer in GTP or GDP state. This led to the polymerization of short and curved filaments, rings, open rings forming clusters, and in the case of BsFtsZ, a novel cylindrical structure of stacked open rings. In vivo, dNAK 4 had almost no effect on the growth of E. coli in liquid culture, in contrast to the strong inhibitory effect observed over B. subtilis growth. The insensitivity of E. coli to this compound is probably related to the impermeability of dNAK 4 to the outer membrane. The low amount of this compound required to inhibit several of the bacterial strains tested and the lack of a cytotoxic effect at the concentrations used, makes dNAK 4 a very good candidate as a starting molecule for the development of a new antibiotic.