Genome-based characterization of two Colombian clinical Providencia rettgeri isolates co-harboring NDM-1, VIM-2, and other ß-lactamases.

BACKGROUND: Providencia rettgeri is a nosocomial pathogen associated with urinary tract infections and related to Healthcare-Associated Infection (HAI). In recent years isolates producing New Delhi Metallo-ß-lactamase (NDM) and other ß-lactamases have been reported that reduce the efficiency of clinical antimicrobial treatments. In this study, we analyzed antibiotic resistance, the presence of resistance genes and the clonal relationship of two P. rettgeri isolates obtained from male patients admitted to the same hospital in Bogotá - Colombia, 2015. RESULTS: Antibiotic susceptibility profile evaluated by the Kirby-Bauer method revealed that both isolates were resistant to third-generation carbapenems and cephalosporins. Whole-genome sequencing (Illumina HiSeq) followed by SPAdes assembling, Prokka annotation in combination with an in-house Python program and resistance gene detection by ResFinder identified the same six ß-lactamase genes in both isolates: blaNDM-1, blaVIM-2, blaCTX-M-15, blaOXA-10, blaCMY-2 and blaTEM-1. Additionally, various resistance genes associated with antibiotic target alteration (arnA, PmrE, PmrF, LpxA, LpxC, gyrB, folP, murA, rpoB, rpsL, tet34) were found and four efflux pumps (RosAB, EmrD, mdtH and cmlA). The additional resistance to gentamicin in one of the two isolates could be explained by a detected SNP in CpxA (Cys191Arg) which is involved in the stress response of the bacterial envelope. Genome BLAST comparison using CGView, the ANI value (99.99%) and the pangenome (using Roary) phylogenetic tree (same clade, small distance) showed high similarity between the isolates. The rMLST analysis indicated that both isolates were typed as rST-61,696, same as the RB151 isolate previously isolated in Bucaramanga, Colombia, 2013, and the FDAARGOS_330 isolate isolated in the USA, 2015. CONCLUSIONS: We report the coexistence of the carbapenemase genes blaNDM-1, and blaVIM-2, together with the ß-lactamase genes blaCTX-M-15, blaOXA-10, blaCMY-2 and blaTEM-1, in P. rettgeri isolates from two patients in Colombia. Whole-genome sequence analysis indicated a circulation of P. rettgeri rST-61,696 strains in America that needs to be investigated further.

Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

Infection by viruses depends on a balance between capsid stability and dynamics. This study investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability and between thermostability and infectivity. In the FMDV capsid, a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essential role for virus infection of most of the 8 tested charged groups is not related to substantial changes in capsid protein expression or processing or in capsid assembly or stability against a thermally induced dissociation into pentamers. However, the positively charged side chains of R2018 and H3141, located at the interpentamer interfaces close to the capsid 2-fold symmetry axes, were found to be critical both for virus infectivity and for keeping the capsid in a state of weak thermostability. A charge-restoring substitution (N2019H) that was repeatedly fixed during amplification of viral genomes carrying deleterious mutations reverted both the lethal and capsid-stabilizing effects of the substitution H3141A, leading to a double mutant virus with close to normal infectivity and thermolability. H3141A and other thermostabilizing substitutions had no detectable effect on capsid resistance to acid-induced dissociation into pentamers. The results suggest that FMDV infectivity requires limited local stability around the 2-fold axes at the interpentamer interfaces of the capsid. The implications for the mechanism of genome uncoating in FMDV and the development of thermostabilized vaccines against foot-and-mouth disease are discussed.IMPORTANCE This study provides novel insights into the little-known structural determinants of the balance between thermal stability and instability in the capsid of foot-and-mouth disease virus and into the relationship between capsid stability and virus infectivity. The results provide new guidelines for the development of thermostabilized empty capsid-based recombinant vaccines against foot-and-mouth disease, one of the economically most important animal diseases worldwide.

Whole-Genome Sequence of a Colombian Acinetobacter baumannii Strain, a Coproducer of OXA-72 and OXA-255-Like Carbapenemases.

Colombian Acinetobacter baumannii strain ST920 was isolated from the sputum of a 68-year-old male patient. This isolate possessed blaOXA-72 and blaOXA-255-like genes. The assembled genome contained 4,104,098 pb and 38.79% G+C content. This is the first case reported of the coproduction (blaOXA-72 and blaOXA-255-like) of carbapenem-hydrolyzing class D ß-lactamases (CHDLs) in Acinetobacter baumannii.

Different functional sensitivity to mutation at intersubunit interfaces involved in consecutive stages of foot-and-mouth disease virus assembly.

Small spherical viruses are paradigms of supramolecular self-assembly. Identifying the specific structural determinants for virus assembly provides guidelines to develop new antiviral drugs or engineer modified viral particles for medical or technological applications. However, very few systematic studies have been carried out so far to identify those chemical groups at interfaces between virus capsid subunits that are important for viral assembly and function. Foot-and-mouth disease virus (FMDV) and other picornaviruses are assembled in a stepwise process in which different protein-protein interfaces are formed: 5 protomeric subunits oligomerize to form a pentameric intermediate, and 12 of these stable pentameric building blocks associate to form a labile capsid. In this study, a systematic mutational analysis revealed that very few amino acid side chains involved in substantial interactions between protomers within each pentamer are individually required for virus infectivity. This result contrasts sharply with the previous finding that most amino acid side chains involved in interactions between pentamers during the next assembly step are individually required for infectivity. The dramatic difference in sensitivity to single mutations between the two types of protein-protein interfaces in FMDV is discussed in terms of possible structural strategies for achieving self-assembly and genome uncoating in the face of diverse selective constraints.

Identification of the structural basis of thermal lability of a virus provides a rationale for improved vaccines.

Virus stability and dynamics play critical roles during infection. Some viruses, including foot-and-mouth disease virus (FMDV), are surprisingly prone to thermal dissociation outside the cell. The structural bases and functional implications of this distinctive trait were essentially unknown. This study (1) uncovers the structural determinants of FMDV thermolability, (2) investigates the relationship between virus thermolability and infectivity, and (3) provides a structure-based rationale for engineering thermostable virus particles to develop improved vaccines and nanocontainers. The results reveal that negatively charged residues close to protein-protein interfaces exert electrostatic repulsions between capsid subunits and mediate the sensitivity of the virion to thermal dissociation, even at neutral pH. Based on these results, a series of fully infectious virions of increased thermostability were engineered by individually removing different carboxylates involved in intersubunit repulsions. The implications for virus biology and the design of thermostable vaccines are discussed.

Lymphotherapy induce an increase of blocking factors and correct infertility problems / La Linfoterapia induce aumento de factores bloqueadores y corrige problemas de infertilidad

This study aimed to confirm the presence of Blocking Factors (BFs) in Mixed Lymphocyte Culture (MLC) from female normal reproducer and sub-fertile rabbit inoculated with two injection of the allogenic lymphotherapy (LIT) to analyze its effect on rate fertility and pregnancy success. The BFs measuring was done intervening MLC with MTT-Formazan non-radioactive technique. It was demonstrated BFs presence in MLC in female rabbit groups. In sub-fertile female reproducers treated with allogenic lymphotherapy a significant increase in the level of FBs after every LIT was observed, as well as a rate fertility increase. Furthermore, it was established that BFs act on cell proliferation inhibiting the MLC of other species, clearly indicating that the inhibit effect of the BFs is inter-specific and no intraspecific as had sustain until now.

Esta investigación se basó en un modelo experimental de origen animal, dirigido a comprobar la existencia de factores bloqueadores (FBs) del cultivo mixto de linfocitos (CML) en grupos de conejas reproductoras normales y subfértiles. A los animales de experimentación se les aplicó dos dosis de Linfoterapia (LIT) alogénica, con el fin de analizar sus efectos en el aumento de la tasa de fertilidad y del éxito gestacional. La medición de los FBs se realizó mediante CML con la técnica no radioactiva MTT-Formazan. Se comprobó la existencia de FBs del CML en todos los grupos de conejas estudiados. En conejas reproductoras subfértiles tratadas con LIT alogénica se observó un incremento significativo de los niveles de los FBs después de cada LIT, así como el aumento en la tasa de fertilidad de las mismas. Además, se estableció que los FBs de proliferación celular actúan inhibiendo el CML de otras especies, lo que indica claramente que el efecto inhibitorio de FBs es interespecífico y no intraespecífico como se ha sostenido hasta ahora.

Comparative evaluation of permissiveness to dengue virus serotype 2 infection in primary rodent macrophages.

Infection with dengue virus presents a broad clinical spectrum, which can range from asymptomatic cases to severe cases that are characterised by haemorrhagic syndrome and/or shock. The reason for such variability remains unknown. This work evaluated the in vitro permissiveness of mouse, rat, hamster and guinea pig macrophages to infection by dengue virus 2 (DENV2). The results established that macrophages derived from the BALB/c mouse strain showed higher permissiveness to DENV2 infection than macrophages from other rodent species, although all rodent species studied had the C820T mutation in the oligoadenylate synthetase 1b gene, indicating no relationship to the different in vitro susceptibilities of mouse cells at this locus. Other molecular mechanisms related to flavivirus susceptibility remain to be explored.

Effects of macromolecular crowding on the inhibition of virus assembly and virus-cell receptor recognition.

Biological fluids contain a very high total concentration of macromolecules that leads to volume exclusion by one molecule to another. Theory and experiment have shown that this condition, termed macromolecular crowding, can have significant effects on molecular recognition. However, the influence of molecular crowding on recognition events involving virus particles, and their inhibition by antiviral compounds, is virtually unexplored. Among these processes, capsid self-assembly during viral morphogenesis and capsid-cell receptor recognition during virus entry into cells are receiving increasing attention as targets for the development of new antiviral drugs. In this study, we have analyzed the effect of macromolecular crowding on the inhibition of these two processes by peptides. Macromolecular crowding led to a significant reduction in the inhibitory activity of: 1), a capsid-binding peptide and a small capsid protein domain that interfere with assembly of the human immunodeficiency virus capsid, and 2), a RGD-containing peptide able to block the interaction between foot-and-mouth disease virus and receptor molecules on the host cell membrane (in this case, the effect was dependent on the conditions used). The results, discussed in the light of macromolecular crowding theory, are relevant for a quantitative understanding of molecular recognition processes during virus infection and its inhibition.

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid lability and confer resistance to acid-dependent uncoating inhibition.

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH(4)Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH(4)Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH(4)Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.

Systematic study of the genetic response of a variable virus to the introduction of deleterious mutations in a functional capsid region.

We have targeted the intersubunit interfaces in the capsid of foot-and-mouth disease virus to investigate the genetic response of a variable virus when individual deleterious mutations are systematically introduced along a functionally defined region of its genome. We had previously found that the individual truncation (by mutation to alanine) of 28 of the 42 amino acid side chains per protomer involved in interactions between capsid pentameric subunits severely impaired infectivity. We have now used viral RNAs individually containing each of those 28 deleterious mutations (or a few others) to carry out a total of 96 transfections of susceptible cells, generally followed by passage(s) of the viral progeny in cell culture. The results revealed a very high frequency of fixation in the capsid of second-site, stereochemically diverse substitutions that compensated for the detrimental effect of primary substitutions at many different positions. Most second-site substitutions occurred at or near the capsid interpentamer interfaces and involved residues that are spatially very close to the originally substituted residue. However, others occurred far from the primary substitution, and even from the interpentamer interfaces. Remarkably, most second-site substitutions involved only a few capsid residues, which acted as "second-site hot spots." Substitutions at these hot spots compensated for the deleterious effects of many different replacements at diverse positions. The remarkable capacity of the virus to respond to the introduction of deleterious mutations in the capsid with the frequent fixation of diverse second-site mutations, and the existence of second-site hot spots, may have important implications for virus evolution.

Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines.

We have rationally engineered foot-and-mouth disease virus to increase its stability against thermal dissociation into subunits without disrupting the many biological functions needed for its infectivity. Amino acid side chains located near the capsid intersubunit interfaces and either predicted or found to be dispensable for infectivity were replaced by others that could establish new disulfide bonds or electrostatic interactions between subunits. Two engineered viruses were normally infectious, genetically stable, and antigenically indistinguishable from the natural virus but showed substantially increased stability against irreversible dissociation. Electrostatic interactions mediated this stabilizing effect. For foot-and-mouth disease virus and other viruses, some evidence had suggested that an increase in virion stability could be linked to an impairment of infectivity. The results of the present study show, in fact, that virion thermostability against dissociation into subunits may not be selectively constrained by functional requirements for infectivity. The thermostable viruses obtained, and others similarly engineered, could be used for the production, using current procedures, of foot-and-mouth disease vaccines that are less dependent on a faultless cold chain. In addition, introduction of those stabilizing mutations in empty (nucleic acid-free) capsids could facilitate the production of infection-risk-free vaccines against the disease, one of the economically most important animal diseases worldwide.

Establecimiento y caracterización de una línea celular derivada de un glioblastoma multiforme / Initiation and characterization of a glioblastoma multiforme derived cell line

Introducción: Las líneas celulares y los cultivos primarios son una excelente herramienta para el estudio de la biología, desarrollo y respuesta a la terapia en tumores cerebrales. Objetivo: Establecer y caracterizar una línea celular derivada de un glioblastoma multiforme como un modelo de estudio in vitro para la extrapolación y aplicación futura en terapia génica. Material y métodos: Se obtuvo una muestra de un paciente con diagnóstico clínico e histopatológico de glioblastoma multiforme, se caracterizó mediante inmunohistoquímica en cortes de tejido y por inmunocitoquímica sobre células cultivadas a partir del tumor desde el inicio del cultivo y durante los seis primeros pases, con dos tipos de marcadores específicos para glía: GFAP (glial fibrillary acidic protein) y S-100 (proteína de unión a calcio). Además, se evaluó la expresión de p53 y Bcl-2, como moduladores de apoptosis. Por último se hizo la caracterización citogenética. Resultados: Histopatológicamente, se confirmó el diagnóstico de glioblastoma multiforme. En los cultivos primarios se encontraron características citomorfológicas propias de un glioblastoma: células fibroblastoides planas, células con escaso citoplasma con 3 ó más procesos y por último bipolares o unipolares. Se encontró una expresión diferencial con los cuatro marcadores, con un patrón de marcaciones a nivel citoplasmático y nuclear a través de los pases estudiados. La línea celular se caracterizó por ser en su mayoría aneuploide con un número modal cromosómico entre 43 y 45, con un gran número de poliploidías (55-102 <4n>, XXYY) y endo-reduplicaciones (end 45, X, -Y). Conclusión: Se estableció una línea celular derivada de un glioblastoma multiforme con un fenotipo estable, con un notable mantenimiento del perfil glial y citogenético.

Introduction: Cell lines and primary cultures are a useful tool for studying basic biology, development and therapy responses in cancer and nervous system tumors. Aim: To establish and characterize a human glioblastoma multiforme (GBM) derived cell line as an in vitro biological model to study nervous system cancer chemotherapy and gene therapy. Materials and methods: A resected tumor piece was obtained from a patient with clinical and histopathological diagnosis of GBM. It was processed to obtain viable cells to culture and histological sections, which were immunostained to glial fibrillary acid protein (GFAP) and S-100 protein (calcium binding protein) and to evaluate expression of apoptosis related proteins p53 and Bcl-2. Finally a cytogenetic evaluation was carried out. Results: Histopathological examination confirmed classic findings of GBM. Typical cytomorphological features of GBM were found in cells of the primary cultures: bipolar or unipolar cells, flat fibroblastoid cells, process-bearing cells with scant cytoplasm and 3 or more processes. It was found a differential expression of the four markers, which had a nuclear and cytoplasmatic staining pattern throughout studied subcultures. Cell line exhibited a high level of aneuploidy with modal chromosomal number between 43-45, with presence of poliploidy (55-102 <4n>, XXYY) and endoreduplication (end 45, X, -Y). Conclusion: It was established a GBM derived cell line with a stable phenotype, maintaining morphological cell and cytogenetic characteristics.

HLA-G: su importancia inmunológica / HLA-G: It´s immunological importance

HLA-G es una molécula clase I no clásica (Ib) del Complejo Mayor de Histocompatibilidad Humano(CMH), adopta siete isoformas, resultado del empalme alternativo de un mismo RNA inmaduro, cuatro seencuentran ligadas a la membrana (HLA-G1,-G2, -G3, -G4) y las otras tres formas son solubles (HLA-G5, -G6, -G7), teniendo en común el dominio extracelular a1. Se expresa de forma selectiva en la interfase maternofetal desempeñando un papel crucial en la inducción de un estado de tolerancia del feto semialogénico por el sistema inmune materno. Está molécula inhibe la citólisis de las NK mediante la interacción con uno o varios receptores inhibidores presentes en las NK (KIR), presenta un limitado polimorfismo, se han descrito15 alelos, 14 de los cuales asignados por la nomenclatura del HLA. El HLA-G está asociado con algunas patologías en la gestación, tales como las infecciones intrauterinas; pre-eclampsia y en aborto recurrente espontáneo. Los estudios van dirigidos a controlar mejor las interacciones HLA-G/células NK en la inducción de un estado de tolerancia inmunitaria en el campo del transplante y la inmunología de los tumores.

Fluorometric cell-ELISA for quantifying rabies infection and heparin inhibition.

The purpose of the present study was to implement a fluorometric method for detecting and quantifying viral antigens in human meduloblastoma cells infected by two types of fixed rabies virus (CVS-MB and CVS-BHK) and a street virus using a cell-enzyme linked immunosorbent assay (cell-ELISA) technique; alkaline phosphatase was used as the antibody-marker enzyme and 4-methyl-umbelliferyl-phosphate as the fluorogenic substrate. The system was used for detecting up to 1:10,000 viral inoculums, followed by evaluating the effect of heparin on infection. Infected cultures were reliably differentiated from their respective negative controls in both assays allowing data to be analysed statistically. As reported in another study, heparin produces strong inhibition when the CVS-BHK viral strain is used for infection; it has thus been suggested that it binds to the neural cell adhesion molecule and could be blocked by using this drug. This fluorometric method is less time-consuming, has increased reproducibility and useful for quantitation of collected data and can therefore be considered as a useful tool for research.

El papel de la linfoterapia en la corrección de problemas de infertilidad de causa inmunológica / Lymphotherapy and fertility

Esta investigación se basa en un modelo experimental de origen animal dirigido a comprobar la existencia de Factores Bloqueadores (FBs) del Cultivo Mixto de Linfocitos (CML) en grupos de conejas reproductoras norma les, y subfértiles a quienes se les aplicó dos dosis de Linfoterapia (LIT) alogénica con el fin de analizar sus efectos en el aumento de la tasa de fertilidad y del éxito gestacional. La medición de los FBs se realizó mediante CML con la técnica no radioactiva MTT-Formazan. Se comprobó la existencia de FBs del CML en todos los grupos de conejas estudiados. En conejas reproductoras subfértiles tratadas con LIT alogénica se observó un incremento significativo de los niveles de los FBs después de cada LIT, así como el aumento en la tasa de fertilidad de las mismas. Además, se estableció que los FBs de proliferación celular actúan inhibiendo el CML de otras especies, lo que indica claramente que el efecto inhibitorio de FBs es interespecífico y no intraespecífico como se ha sostenido hasta ahora