Uncovering Surface Penetration by Enterococci From Urinary Tract Infection Patients.

IMPORTANCE: The relationship between Enterococcus faecalis vaginal colonization and urinary tract infections (UTIs) remains uncertain. OBJECTIVE: We aimed to evaluate the surface invasion capability of E faecalis isolates from patients with and without UTIs as a potential readout of pathogenicity. STUDY DESIGN: Participants were females from urogynecology clinics, comprising symptomatic UTI and asymptomatic non-UTI patients, categorized by the presence or absence of E faecalis-positive cultures identified via standard urine culture techniques. Vaginal and urine samples from patients were plated on enterococci selective medium, and E faecalis isolates detected in both cohorts were species specific identified using 16S rRNA sequencing. Clinical isolates were inoculated on semisolid media, and both external colonies and underneath colony prints formed by agar-penetrating enterococci were imaged. External growth and invasiveness were quantified by determining colony-forming units of the noninvading and agar-penetrating cells and compared with the E faecalis OG1RF. RESULTS: We selected E faecalis isolates from urine and vaginal samples of 4 patients with and 4 patients without UTIs. Assays demonstrated that most isolates formed similarly sized external colonies with comparable colony-forming unit. Surface invasion differed across patients and isolation sites compared with OG1RF. The vaginal isolate from UTI patient 1, who had the most recurrences, exhibited significantly greater agar-invading capacity compared with OG1RF. CONCLUSIONS: Our pilot study indicates that ex vivo invasion assays may unveil virulence traits in E faecalis from UTI patients. Enhanced enterococcal surface penetration could increase urogenital invasion risk. Further research is needed to correlate penetration with disease severity in a larger patient group.

Transgelin 2 guards T cell lipid metabolic programming and anti-tumor function.

Mounting effective immunity against pathogens and tumors relies on the successful metabolic programming of T cells by extracellular fatty acids1-3. During this process, fatty-acid-binding protein 5 (FABP5) imports lipids that fuel mitochondrial respiration and sustain the bioenergetic requirements of protective CD8+ T cells4,5. Importantly, however, the mechanisms governing this crucial immunometabolic axis remain unexplored. Here we report that the cytoskeletal organizer Transgelin 2 (TAGLN2) is necessary for optimal CD8+ T cell fatty acid uptake, mitochondrial respiration, and anti-cancer function. We found that TAGLN2 interacts with FABP5, enabling the surface localization of this lipid importer on activated CD8+ T cells. Analysis of ovarian cancer specimens revealed that endoplasmic reticulum (ER) stress responses elicited by the tumor microenvironment repress TAGLN2 in infiltrating CD8+ T cells, enforcing their dysfunctional state. Restoring TAGLN2 expression in ER-stressed CD8+ T cells bolstered their lipid uptake, mitochondrial respiration, and cytotoxic capacity. Accordingly, chimeric antigen receptor T cells overexpressing TAGLN2 bypassed the detrimental effects of tumor-induced ER stress and demonstrated superior therapeutic efficacy in mice with metastatic ovarian cancer. Our study unveils the role of cytoskeletal TAGLN2 in T cell lipid metabolism and highlights the potential to enhance cellular immunotherapy in solid malignancies by preserving the TAGLN2-FABP5 axis.

Remodeling of the Enterococcal Cell Envelope during Surface Penetration Promotes Intrinsic Resistance to Stress.

Enterococcus faecalis is a normal commensal of the human gastrointestinal tract (GIT). However, upon disruption of gut homeostasis, this nonmotile bacterium can egress from its natural niche and spread to distal organs. While this translocation process can lead to life-threatening systemic infections, the underlying mechanisms remain largely unexplored. Our prior work showed that E. faecalis migration across diverse surfaces requires the formation of matrix-covered multicellular aggregates and the synthesis of exopolysaccharides, but how enterococcal cells are reprogrammed during this process is unknown. Whether surface penetration endows E. faecalis with adaptive advantages is also uncertain. Here, we report that surface penetration promotes the generation of a metabolically and phenotypically distinct E. faecalis population with an enhanced capacity to endure various forms of extracellular stress. Surface-invading enterococci demonstrated major ultrastructural alterations in their cell envelope characterized by increased membrane glycolipid content. These changes were accompanied by marked induction of specific transcriptional programs enhancing cell envelope biogenesis and glycolipid metabolism. Notably, the surface-invading population demonstrated superior tolerance to membrane-damaging antimicrobials, including daptomycin and ß-defensins produced by epithelial cells. Genetic mutations impairing glycolipid biosynthesis sensitized E. faecalis to envelope stressors and reduced the ability of this bacterium to penetrate semisolid surfaces and translocate through human intestinal epithelial cell monolayers. Our study reveals that surface penetration induces distinct transcriptional, metabolic, and ultrastructural changes that equip E. faecalis with enhanced capacity to resist external stressors and thrive in its surrounding environment. IMPORTANCE Enterococcus faecalis inhabits the GIT of multiple organisms, where its establishment could be mediated by the formation of biofilm-like aggregates. In susceptible individuals, this bacterium can overgrow and breach intestinal barriers, a process that may lead to lethal systemic infections. While the formation of multicellular aggregates promotes E. faecalis migration across surfaces, little is known about the metabolic and physiological states of the enterococci encased in these surface-penetrating structures. The present study reveals that E. faecalis cells capable of migrating through semisolid surfaces genetically reprogram their metabolism toward increased cell envelope and glycolipid biogenesis, which confers superior tolerance to membrane-damaging agents. E. faecalis's success as a pathobiont depends on its antimicrobial resistance, as well as on its rapid adaptability to overcome multiple environmental challenges. Thus, targeting adaptive genetic and/or metabolic pathways induced during E. faecalis surface penetration may be useful to better confront infections by this bacterium in the clinic.

Exopolysaccharide-mediated surface penetration as new virulence trait in Enterococcus faecalis.

Enterococcus faecalis is a commensal bacterium that normally inhabits the gastrointestinal tract of humans. This non-motile microorganism can also cause lethal infections in other organs by penetrating and breaching the intestinal barrier. However, the precise molecular mechanisms enabling E. faecalis movement and translocation across epithelial barriers remain incompletely characterized. We recently reported that E. faecalis utilizes the RpiA-GlnA-EpaX metabolic axis to generate ß-1,6-linked poly-N-acetylglucosamine (polyGlcNAc)-containing exopolymers that are necessary for its optimal migration into semisolid surfaces and efficient translocation through human epithelial cell monolayers. These findings provide new evidence indicating that non-motile bacterial pathogens can exploit carbohydrate metabolism to penetrate surfaces. Hence, targeting this process might represent a new strategy to more effectively control systemic infections by E. faecalis.

PolyGlcNAc-containing exopolymers enable surface penetration by non-motile Enterococcus faecalis.

Bacterial pathogens have evolved strategies that enable them to invade tissues and spread within the host. Enterococcus faecalis is a leading cause of local and disseminated multidrug-resistant hospital infections, but the molecular mechanisms used by this non-motile bacterium to penetrate surfaces and translocate through tissues remain largely unexplored. Here we present experimental evidence indicating that E. faecalis generates exopolysaccharides containing ß-1,6-linked poly-N-acetylglucosamine (polyGlcNAc) as a mechanism to successfully penetrate semisolid surfaces and translocate through human epithelial cell monolayers. Genetic screening and molecular analyses of mutant strains identified glnA, rpiA and epaX as genes critically required for optimal E. faecalis penetration and translocation. Mechanistically, GlnA and RpiA cooperated to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that was utilized by EpaX to synthesize polyGlcNAc-containing polymers. Notably, exogenous supplementation with polymeric N-acetylglucosamine (PNAG) restored surface penetration by E. faecalis mutants devoid of EpaX. Our study uncovers an unexpected mechanism whereby the RpiA-GlnA-EpaX metabolic axis enables production of polyGlcNAc-containing polysaccharides that endow E. faecalis with the ability to penetrate surfaces. Hence, targeting carbohydrate metabolism or inhibiting biosynthesis of polyGlcNAc-containing exopolymers may represent a new strategy to more effectively confront enterococcal infections in the clinic.

Isolation of PCR-quality Genomic DNA from Soils Impacted with Extra Heavy Crude Oil.

For the study of microbial communities in samples of soils impacted with extra heavy crude oil, it is necessary to perform molecular analyses. Due to the difficulty of oil matrix handling, there are very few protocols reported in writing. Also, one can only observe a very low concentration of DNA. That's why it is required to have an effective protocol to conduct studies in this type of matrix. This protocol includes steps of cell lysis by saline buffer with ionic/non-ionic detergents, and enzymatic digestion with lysozyme and proteases, complemented with organic extraction and alcohol precipitation. Additionally, it requires purification to eliminate the inhibitory substances of the extract that cause PCR inhibition. The method of DNA extraction proposed in this study is easy to handle and low cost. It allows the extraction of DNA from different bacteria and fungi, associated with soil contaminated with extra heavy crude.

A cluster of KPC-2 and VIM-2-producing Klebsiella pneumoniae ST833 isolates from the pediatric service of a Venezuelan Hospital.

BACKGROUND: Klebsiella pneumoniae is a bacterial pathogen that has developed resistance to multiple antibiotics and is a major cause of nosocomial infections worldwide. Carbapenemase-producing Klebsiella pneumoniae have been isolated in many hospitals in Venezuela, but they have not been well-studied. The aim of this study was to characterize carbapenem-resistant Klebsiella pneumoniae isolates from the pediatric service of a hospital located in Anzoategui State, in the eastern part of Venezuela. METHODS: Nineteen Klebsiella pneumoniae strains isolated in the hospital from April to July 2014 were evaluated phenotypically and molecularly for the presence of carbapenemases blaKPC, blaIMP and blaVIM. Molecular epidemiology was performed with Repetitive Extragenic Palindromic-PCR (REP-PCR) and Multilocus Sequence Typing (MLST). They were also studied for phenotypic and molecular resistance to a quaternary ammonium compound (QAC) disinfectant. RESULTS: All 19 isolates contained both bla VIM-2 and bla KPC-2 genes, and the bla KPC-2 gene was associated with Tn4401b. All isolates were phenotypically sensitive to QACs and contained qacΔE and addA2 genes typical of class 1 integrons. Analysis by REP-PCR and MLST showed that all isolates had identical profiles characteristic of sequence type ST833. CONCLUSION: All 19 strains are bla VIM-2 and bla KPC-2-producing ST833 K. pneumoniae sensitive to QACs. This analysis may help to understand the routes of dissemination and confirms that QAC disinfectants can be used to help control their spread.

Evaluación de la resistencia a agentes desinfectantes de bacterias aisladas de ambientes naturales / Evaluation of resistance to disinfectant agents of bacteria isolated from natural environments

Los antisépticos y desinfectantes son utilizados extensivamente para la prevención, vigilancia y control de las infecciones. Entre los más utilizados se encuentran los compuestos catiónicos de amonio cuaternario (QACs). El uso y abuso cotidiano de estos compuestos ha conllevado a la selección de cepas bacterianas resistentes. En este estudio, nos propusimos evaluar la resistencia a agentes desinfectantes en cepas bacterianas, multiresistentes a los antibióticos de uso frecuente, aisladas de ambientes naturales, y bacterias aisladas de pacientes hospitalizados. Se utilizó la prueba de suspensión cuantitativa para evaluar los niveles de resistencia, y ensayos de conjugación bacteriana para examinar la posible asociación de los determinantes de resistencia a moléculas plasmídicas transferibles. Los microorganismos gramnegativos, como Pseudomonas aeruginosa, resultaron ser los más resistentes. Las cepas ambientales presentaron niveles mayores de resistencia en comparación con las hospitalarias. Se obtuvieron transconjugantes con niveles de resistencia semejantes a las respectivas cepas donantes, indicando que estos determinantes de resistencia a desinfectantes están codificados en plásmidos, y cuyo análisis por patrones de restricción demostró que existen, al menos, dos moléculas plasmídicas diseminadas entre las cepas del mismo hábitat. Nuestros resultados representan un aporte que permitirá implementar medidas más exitosas para evitar la diseminación de estas bacterias resistentes.

Antiseptics and disinfectants are widely used for prevention, surveillance and control of infections. Among those most used are quaternary ammonium cationic compounds (QACs). The daily use and abuse of these compounds has resulted in the selection of resistant bacterial strains. In this study we decided to evaluate the resistance to disinfectant agents of bacterial strains multiresistant to frequently used antibiotics, both isolated from natural environments and isolated from hospital patients. The quantitative suspension test was used to evaluate resistance levels, and bacterial conjugation assays to examine the possible association of resistance determinants to transferable plasmid molecules. Gram negative microorganisms such as Pseudomonas aeruginosa appeared to be the most resistant. Environmental strains showed higher resistance levels as compared with hospital strains. We obtained transconjugants with resistance levels similar to those of the respective donor strain, indicating that these determinants of resistance to disinfectants are coded in plasmids; when analyzed by restriction patterns they showed that there were at least two plasmid molecules disseminated among the strains of the same habitat. Our results represent a contribution that will allow to implement more successful measures to avoid the dissemination of these resistant bacteria.