Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Proteomics analysis reveals a role for E. coli polyphosphate kinase in membrane structure and polymyxin resistance during starvation.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and ∆ppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of required building blocks. From our dataset, we were particularly interested in Arn and EptA proteins, which were downregulated in ∆ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in ∆ppk cells stems from a failure to induce the BasS/BasR two-component system during starvation. We also show that ∆ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Increased Detection of Carbapenemase-Producing Enterobacterales Bacteria in Latin America and the Caribbean during the COVID-19 Pandemic.

During 2020-2021, countries in Latin America and the Caribbean reported clinical emergence of carbapenemase-producing Enterobacterales that had not been previously characterized locally, increased prevalence of carbapenemases that had previously been detected, and co-production of multiple carbapenemases in some isolates. These increases were likely fueled by changes related to the COVID-19 pandemic, including empirical antibiotic use for potential COVID-19-related bacterial infections and healthcare limitations resulting from the rapid rise in COVID-19 cases. Strengthening antimicrobial resistance surveillance, epidemiologic research, and infection prevention and control programs and antimicrobial stewardship in clinical settings can help prevent emergence and transmission of carbapenemase-producing Enterobacterales.

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

Acinetobactin-Mediated Inhibition of Commensal Bacteria by Acinetobacter baumannii.

Acinetobacter baumannii is an important hospital-associated pathogen that causes antibiotic resistant infections and reoccurring hospital outbreaks. A. baumannii's ability to asymptomatically colonize patients is a risk factor for infection and exacerbates its spread. However, there is little information describing the mechanisms it employs to colonize patients. A. baumannii often colonizes the upper respiratory tract and skin. Antibiotic use is a risk factor for colonization and infection suggesting that A. baumannii likely competes with commensal bacteria to establish a niche. To begin to investigate this possibility, we cocultured A. baumannii and commensal bacteria of the upper respiratory tract and skin. In conditions that mimic iron starvation experienced in the host, we observed that A. baumannii inhibits Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and Corynebacterium striatum. Then using an ordered transposon library screen we identified the A. baumannii siderophore acinetobactin as the causative agent of the inhibition phenotype. Using mass spectrometry, we show that acinetobactin is released from A. baumannii under our coculture conditions and that purified acinetobactin can inhibit C. striatum and S. hominis. Together our data suggest that acinetobactin may provide a competitive advantage for A. baumannii over some respiratory track and skin commensal bacteria and possibly support its ability to colonize patients. IMPORTANCE The ability of Acinetobacter baumannii to asymptomatically colonize patients is a risk factor for infection and exacerbates its clinical spread. However, there is minimal information describing how A. baumannii asymptomatically colonizes patients. Here we provide evidence that A. baumannii can inhibit the growth of many skin and upper respiratory commensal bacteria through iron competition and identify acinetobactin as the molecule supporting its nutritional advantage. Outcompeting endogenous commensals through iron competition may support the ability of A. baumannii to colonize and spread among patients.

A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae.

Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

Homeoviscous Adaptation of the Acinetobacter baumannii Outer Membrane: Alteration of Lipooligosaccharide Structure during Cold Stress.

To maintain optimal membrane dynamics, cells from all domains of life must acclimate to various environmental signals in a process referred to as homeoviscous adaptation. Alteration of the lipid composition is critical for maintaining membrane fluidity, permeability of the lipid bilayer, and protein function under diverse conditions. It is well documented, for example, that glycerophospholipid content varies substantially in both Gram-negative and Gram-positive bacteria with changes in growth temperature. However, in the case of Gram-negative bacteria, far less is known concerning structural changes in lipopolysaccharide (LPS) or lipooligosaccharide (LOS) during temperature shifts. LPS/LOS is anchored at the cell surface by the highly conserved lipid A domain and localized in the outer leaflet of the outer membrane. Here, we identified a novel acyltransferase, termed LpxS, involved in the synthesis of the lipid A domain of Acinetobacter baumannii. A. baumannii is a significant, multidrug-resistant, opportunistic pathogen that is particularly difficult to clear from health care settings because of its ability to survive under diverse conditions. LpxS transfers an octanoate (C8:0) fatty acid, the shortest known secondary acyl chain reported to date, replacing a C12:0 fatty acid at the 2' position of lipid A. Expression of LpxS was highly upregulated under cold conditions and likely increases membrane fluidity. Furthermore, incorporation of a C8:0 acyl chain under cold conditions increased the effectiveness of the outer membrane permeability barrier. LpxS orthologs are found in several Acinetobacter species and may represent a common mechanism for adaptation to cold temperatures in these organisms. IMPORTANCE To maintain cellular fitness, the composition of biological membranes must change in response to shifts in temperature or other stresses. This process, known as homeoviscous adaptation, allows for maintenance of optimal fluidity and membrane permeability. Here, we describe an enzyme that alters the fatty acid content of A. baumannii LOS, a major structural feature and key component of the bacterial outer membrane. Although much is known regarding how glycerophospholipids are altered during temperature shifts, our understanding of LOS or LPS alterations under these conditions is lacking. Our work identifies a cold adaptation mechanism in A. baumannii, a highly adaptable and multidrug-resistant pathogen.

Teenagers and Precision Psychiatry: A Window of Opportunity.

OBJECTIVE: Precision medicine raises hope for translating genetic-based knowledge about psychiatric risks into mental health benefits by motivating health-related, risk-reducing behaviors. Teenagers (ages 14-17) are an important age-group to engage in preventive efforts but, their views about psychiatric genetics are understudied. METHOD: An online survey with a nationally representative sample of teenagers (n = 417) was conducted. Participants were randomly assigned to receive 1 of 2 handouts, 1 emphasizing the genetic underpinnings of psychiatric conditions; the other agency-oriented and focusing on gene-environment interactions. Survey questions queried their views about behavioral changes in response to psychiatric genetic risk information and expressed willingness to undertake them. Participants' decision-making characteristics (i.e., self-efficacy, empowerment, intolerance of uncertainty, and sensation-seeking) were assessed at baseline. RESULTS: Teenagers strongly valued the information provided and its potential usefulness for their mental health. Information about psychiatric genetics alone impacted views about the causes of mental illness. Contrary to our hypothesis, the type of handout did not impact participants' expressed willingness to make behavioral changes to reduce their risk of developing a psychiatric condition, but their sense of empowerment played a key role in their responses. CONCLUSION: Educating teenagers about gene-environment interactions may help facilitate the translational efforts of precision psychiatry. Research with teenagers across racial/ethnic groups, especially those with family histories, is needed to better understand the factors that impact teenagers' empowerment in psychiatric genomic settings and to identify measures, including the best enablers of empowerment (e.g., educators, parents), which would allow them to reap the benefits of precision psychiatry.

El papel de las pruebas diagnósticas en el manejo de la pandemia COVID-19: un enfoque desde el laboratorio clínico / The role of diagnostic tests in the management of the COVID-19 pandemic: an approach from the clinical laboratory

A finales del 2019 una crisis sanitaria se desató a nivel mundial debido a la propagación del nuevo virus SARSCoV-2 causante de la enfermedad COVID-19. En pocos meses el virus llegó a más de 120 países, causando cerca de 19.5 millones de casos y 725,000 muertes alrededor del mundo. La sintomatología de la enfermedad incluye fiebre, tos, cefalea, dolor de garganta, dificultad respiratoria, fatiga y mialgia. El espectro de la enfermedad puede ir desde los pacientes asintomáticos o leves (la gran mayoría de los casos) hasta aquellos que evolucionan a condiciones que amenazan la vida como el síndrome de dificultad respiratoria aguda, neumonía severa o fallo multiorgánico, principalmente en personas mayores y con comorbilidades. En Guatemala la letalidad es del 3.9%. El diagnóstico de laboratorio clínico juega un papel importante en el control de la pandemia. El diagnóstico se basa en la detección del virus en hisopados nasofaríngeos a través de técnicas moleculares de amplificación de ácidos nucleicos. Otras técnicas de laboratorio resultan importantes para conocer la dinámica de la enfermedad, entre estas se incluyen pruebas para detectar antígeno del virus en secreciones respiratorias y pruebas serológicas para detectar y medir los anticuerpos generados contra el virus.

At the end of 2019, a health crisis broke out worldwide due to the spread of the new SARS-CoV-2 virus that causes the COVID-19 disease. In just a few months the virus reached more than 120 countries, causing about 19.5 million cases and 725,000 deaths around the world. Symptoms of the disease include fever, cough, headache, sore throat, shortness of breath, fatigue, and myalgia. The spectrum of the disease can range from asymptomatic or mild patients (the vast majority of cases) to those who evolve to life-threatening conditions such as acute respiratory distress syndrome, severe pneumonia or multiple organ failure, mainly in older people and people with comorbidities. In Guatemala, the fatality rate is 3.9%. Clinical laboratory diagnosis plays an important role in controlling the pandemic. The diagnosis is based on the detection of the virus in nasopharyngeal swabs through molecular nucleic acid amplification techniques. Other laboratory techniques are important to understand the dynamics of the disease and include tests to detect virus antigen in respiratory secretions and serological tests to detect and measure antibodies generated against the virus.

CrrB Positively Regulates High-Level Polymyxin Resistance and Virulence in Klebsiella pneumoniae.

Polymyxin resistance (PR) threatens the treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. PR frequently arises through chemical modification of the lipid A portion of lipopolysaccharide. Various mutations are implicated in PR, including in three two-component systems-CrrA/B, PmrA/B, and PhoP/Q-and the negative regulator MgrB. Few have been functionally validated. Therefore, here we adapt a CRISPR-Cas9 system to CRKP to elucidate how mutations in clinical CRKP isolates induce PR. We demonstrate that CrrB is a positive regulator of PR, and common clinical mutations lead to the addition of both 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosophethanolamine (pEtN) to lipid A, inducing notably higher polymyxin minimum inhibitory concentrations than mgrB disruption. Additionally, crrB mutations cause a significant virulence increase at a fitness cost, partially from activation of the pentose phosphate pathway. Our data demonstrate the importance of CrrB in high-level PR and establish important differences across crrB alleles in balancing resistance with fitness and virulence.

Unsaturation Elements and Other Modifications of Phospholipids in Bacteria: New Insight from Ultraviolet Photodissociation Mass Spectrometry.

Glycerophospholipids (GPLs), one of the main components of bacterial cell membranes, exhibit high levels of structural complexity that are directly correlated with biophysical membrane properties such as permeability and fluidity. This structural complexity arises from the substantial variability in the individual GPL structural components such as the acyl chain length and headgroup type and is further amplified by the presence of modifications such as double bonds and cyclopropane rings. Here we use liquid chromatography coupled to high-resolution and high-mass-accuracy ultraviolet photodissociation mass spectrometry for the most in-depth study of bacterial GPL modifications to date. In doing so, we unravel a diverse array of unexplored GPL modifications, ranging from acyl chain hydroxyl groups to novel headgroup structures. Along with characterizing these modifications, we elucidate general trends in bacterial GPL unsaturation elements and thus aim to decipher some of the biochemical pathways of unsaturation incorporation in bacterial GPLs. Finally, we discover aminoacyl-PGs not only in Gram-positive bacteria but also in Gram-negative C. jejuni, advancing our knowledge of the methods of surface charge modulation that Gram-negative organisms may adopt for antibiotic resistance.

Genetic Manipulation of Wild Human Gut Bacteroides.

Bacteroides is one of the most prominent genera in the human gut microbiome, and study of this bacterial group provides insights into gut microbial ecology and pathogenesis. In this report, we introduce a negative selection system for rapid and efficient allelic exchange in wild Bacteroides species that does not require any alterations to the genetic background or a nutritionally defined culture medium. In this approach, dual antibacterial effectors normally delivered via type VI secretion are targeted to the bacterial periplasm under the control of tightly regulated anhydrotetracycline (aTC)-inducible promoters. Introduction of aTC selects for recombination events producing the desired genetic modification, and the dual effector design allows for broad applicability across strains that may have immunity to one counterselection effector. We demonstrate the utility of this approach across 21 human gut Bacteroides isolates representing diverse species, including strains isolated directly from human donors. We use this system to establish that antimicrobial peptide resistance in Bacteroides vulgatus is determined by the product of a gene that is not included in the genomes of previously genetically tractable members of the human gut microbiome.IMPORTANCE Human gut Bacteroides species exhibit strain-level differences in their physiology, ecology, and impact on human health and disease. However, existing approaches for genetic manipulation generally require construction of genetically modified parental strains for each microbe of interest or defined medium formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of diverse wild-type Bacteroides species from the human gut. This system enables genetic investigation of members of human and animal microbiomes beyond existing model organisms.

A Whole-Cell Screen Identifies Small Bioactives That Synergize with Polymyxin and Exhibit Antimicrobial Activities against Multidrug-Resistant Bacteria.

The threat of diminished antibiotic discovery has global health care in crisis. In the United States, it is estimated each year that over 2 million bacterial infections are resistant to first-line antibiotic treatments and cost in excess of 20 billion dollars. Many of these cases result from infection with the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which are multidrug-resistant bacteria that often cause community- and hospital-acquired infections in both healthy and immunocompromised patients. Physicians have turned to last-resort antibiotics like polymyxins to tackle these pathogens, and as a consequence, polymyxin resistance has emerged and is spreading. Barring the discovery of new antibiotics, another route to successfully mitigate polymyxin resistance is to identify compounds that can complement the existing arsenal of antibiotics. We recently designed and performed a large-scale robotic screen to identify 43 bioactive compounds that act synergistically with polymyxin B to inhibit the growth of polymyxin-resistant Escherichia coli Of these 43 compounds, 5 lead compounds were identified and characterized using various Gram-negative bacterial organisms to better assess their synergistic activity with polymyxin. Several of these compounds reduce polymyxin to an MIC of <2 µg/ml against polymyxin-resistant and polymyxin-heteroresistant Gram-negative pathogens. Likewise, four of these compounds exhibit antimicrobial activity against Gram-positive bacteria, one of which rapidly eradicated methicillin-resistant Staphylococcus aureus We present multiple first-generation (i.e., not yet optimized) compounds that warrant further investigation and optimization, since they can act both synergistically with polymyxin and also as lone antimicrobials for combating ESKAPE pathogens.

A DedA Family Membrane Protein Is Required for Burkholderia thailandensis Colistin Resistance.

Colistin is a "last resort" antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations (MIC) often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in Burkholderia thailandensis (DbcA; DedA of Burkholderia required for colistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of ΔdbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis ΔdbcA was observed by expression of dbcA, E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis ΔdbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species.

Safinamide in Clinical Practice: A Spanish Multicenter Cohort Study.

Background: Safinamide is an approved drug for the treatment of motor fluctuations of Parkinson's Disease (PD) patients with a potential benefit on non-motor symptoms (NMS). Methods: A retrospective multicenter cohort study was conducted, in which the clinical effect of safinamide on both motor and NMS was assessed by the Clinical Global Impression of Change scale. Furthermore, we assessed the appearance of adverse events (AEs) and its effect on dyskinesia, that were also recorded in non-fluctuating PD patients and in those previously treated with rasagiline. Results: We included 213 PD patients who received safinamide in addition to their regular levodopa therapy. Thirty-five withdrew prematurely from safinamide, mainly because of AEs. Out of 178, clinical improvement on motor and NMS was found in 76.4% and 26.2%, respectively. A total of 44 reported AEs of mild intensity. We did not find a difference concerning the clinical benefit or AEs when comparing either patients who had or had not been taking Monoamine Oxidase B Inhibitor (MAOB-I) previously or between patients with and without motor complications. Conclusions: Safinamide is an effective and safe add-on to levodopa drug for PD patients. Moreover, safinamide could elicit an additional clinical improvement in PD patients previously treated with other MAOB-I and in non- fluctuating patients with suboptimal motor control.

Loss of a Cardiolipin Synthase in Helicobacter pylori G27 Blocks Flagellum Assembly.

Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella.IMPORTANCEH. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.

Infección Por El Virus Linfotrópico De Células T Humano HTLV-1 Y Paraparesis Espástica Tropical En Ecuador: Paradigma De Enfermedad Tropical Desatendida / Human T-lymphotrophic Virus Infection (HTLV-1) And Tropical spastic Paraparesis: Paradigms Of An Overlooked Tropical Disease

Resumen La infección por el virus linfotrópico de células T humano de tipo 1 (HTLV-1) es considerada endémica en América del Sur. Desde hace más de dos décadas existen reportes de casos clínicos de mielopatías asociadas a infección por el retrovirus HTLV-1 en pacientes de Ecuador, patología denominada paraparesis espástica tropical. Sólo hay dos estudios de seroprevalencia de HTLV-1 en Ecuador, ambos con tamaños muestrales pequeños pero en ambos casos indicativos de una alta prevalencia de HTLV-1, al menos comunidades afrodescendientes e indígenas de Ecuador. Pese a ello, y a diferencia de países vecinos, no se realiza tamizaje de bancos de sangre y órganos por parte de las autoridades sanitarias competentes. Por tanto, esta infección y las patologías asociadas a la misma (como la paraparesis espástica tropical) se encontrarían desatendidas y sub diagnosticadas. En definitiva, urge desarrollar estudios epidemiológicos de prevalencia a nivel del país e implementar estrategias de control y prevención para evitar la diseminación de esta infección.

Abstract The human T cell lymphotropic virus (HTLV-1) infection is considered endemic in South America. For more than two decades clinical cases of HTLV-1 associated mielopaties have been reported in Ecuador, pathology known as tropical spastic paraparesis. There is only two studies about HTLV-1 seroprevalence in Ecuador, both of them with small sample size but showing a high prevalence of HTLV-1 infection, at least for afrodescendants and indigenous communities. Howewer, there is not a currently screening for blood and organ banks carried out by ecuadorian public health authorities. This scenario makes HTLV-1 infection and associated pathologies (like tropical spastic paraparesis) neglected diseases in Ecuador. More epidemiological studies need to be implemented in order to develope control and prevention strategies in the country.

A high prevalence of human T-lymphotropic virus (HTLV 1/2) infection among Afro-descendants, Esmeraldas province, Ecuador - need for the implementation of surveys and control programs.

Background: Infection with the Human T-cell lymphotropic virus (HTLV) type 1 and 2 is endemic in South America. Although there are reports of the presence of these retroviruses in Ecuador, the infection is usually neglected in healthcare settings and by public health authorities, and no actualized prevalence data are available. Objectives: This cross-sectional study aimed to estimate, using for the first time to our knowledge in Ecuador immunological and molecular biology methodologies, the prevalence of HTLV-1/2 infection in asymptomatic individuals from a potentially high-risk population of Afro-Ecuadorians in the Province of Esmeraldas. Study design: Two hundred and twenty-seven plasma samples from asymptomatic individuals were analyzed for the detection of the HTLV-1/2 virus with three methods: a commercial ELISA, an indirect immunofluorescence (IF) assay, and a nested-PCR. Results: The overall prevalence of HTLV-1/2 antibodies in this population, as determined with an ELISA, was 11.0%. Both the IF assay and the nested-PCR confirmed a prevalence of 3.5%. Conclusion: The high prevalence of HTLV-1/2 infection among the Afro-Ecuadorian population of Esmeraldas Province shows the need of the implementation of control and prevention interventions to overcome the further dissemination of the infection. To define the real problem of HTLV-1/2 infection in Ecuador, more prevalence studies have to be undertaken in other Afro-Ecuadorian populations and high-risk populations like the indigenous population of the Andes Mountains and the tropical Amazon region.

Isolation of Lipid Cell Envelope Components from Acinetobacter baumannii.

With the increasing occurrence of antibiotic resistance among Acinetobacter sp., the race is on for researchers to not only isolate resistant isolates but also utilize basic and applied microbiological techniques to study mechanisms of resistance. For many antibiotics, the limit of efficacy against Gram-negative bacteria is dependent on its ability to permeate the outer membrane and access its target. As such, it is critical that researchers be able to isolate and analyze the lipid components of the cell envelope from any number of Acinetobacter sp. that are either resistant or sensitive to antibiotics of interest. The following chapter provides in-depth protocols to confirm the presence or absence of lipooligosaccharide (LOS) in Acinetobacter sp., isolate lipid A, and glycerophospholipids and analyze them using qualitative (mass spectrometry) and semiquantitative (thin-layer chromatography) methods.

Cx43 overexpression in osteocytes prevents osteocyte apoptosis and preserves cortical bone quality in aging mice.

Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age-related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1-8kb promoter (Cx43OT mice) attenuates the skeletal cortical, but not trabecular bone phenotype of aged, 14-month-old mice. The percentage of Cx43-expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43 positive osteoblasts remained similar to wild type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared to skeletally mature, 6-month-old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age-matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers, compared to skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared to young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces, and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age-induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength.