Resistencia plasmídica a colistin por el gen mcr-1 en Enterobacteriaceae en Paraguay / Plasmid-Mediated Colistin Resistance Gene mcr-1 in Enterobacteriaceae in Paraguay

La resistencia a las polimixinas mediada por plásmidos (gen mcr-1) representa una amenaza para la salud pública, puesto que colistina es utilizada en la práctica médica como una de las últimas alternativas para el tratamiento de gérmenes multiresistentes. Este estudio describe la circulaciónde cepas de Enterobacterias que portan este gen de resistencia, aisladas de pacientes hospitalizados, así como también de la comunidad. Los hallazgos de la Red de Vigilancia de la Resistencia a los Antimicrobianos-Paraguay fueron de casi el 5 % (4,7) en cepas remitidas con criterio de sospecha, siendo las especies involucradas Escherichiacoli, Klebsiella pneumoniae y Salmonella Schwarzengrund. Además, por métodos moleculares se confirmaron en todas ellas la portación de otros genes de resistencia (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) asociados al mcr-1. Palabras claves: Enterobacterias, resistencia, colistina, mcr-1.

Resistance to polymyxins mediated by plasmids (mcr-1 gene) represents a threat to public health, since colistin is used in medical practice, as one of the last alternatives, for the treatment of multi-resistant germs. This study describes the circulation of strains of Enterobacteria that carry this resistance gene, isolated from hospitalized patients, as well as from the community. The findings of the Red de Vigilancia de la Resistencia a los Antimicrobianos­Paraguay were almost 5% (4.7) in strains submitted with suspicion criteria; the species involved being Escherichia coli, Klebsiella pneumoniae and Salmonella Schwarzengrund. In addition, molecular methods confirmed in all of them the carrying of other resistance genes (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) associated with mcr-1. Key words: Enterobacteria, resistance, colistin, mcr-1.

Primer aislamiento de Salmonella Javiana con portación de KPC-2 en Paraguay / First isolation of Salmonella Javiana with KPC-2 in Paraguay

Varios mecanismos de resistencia a diversos agentes antimicrobianos que se han incorporado al arsenal terapéutico, han emergido en los últimos años, destacando la aparición de carbapenemasas en enterobacterias, siendo Klebsiella pneumoniae carbapenemasa (KPC) una de las más importantes, identificada originalmente en los Estados Unidos en 1996. En Paraguay, los primeros aislamientos de cepas portadoras de carbapenemasa tipo KPC se obtuvieron en septiembre del año 2009, a partir del cual se confirmaron la presencia en varias especies de enterobacterias, siendo Klebsiella pneumoniae la de mayor prevalencia entre ellas. Según datos del centro de referencia, Salmonella entérica serotipo Typhimurium es el serovar más común identificado en muestras de heces hasta la fecha, y el serotipo Javiana el cuarto. En este informe se describe el primer aislamiento resistente a carbapenemes por carbapenemasa del tipo KPC-2 en Salmonella entérica serotipo Javiana aislado en febrero de 2015 de una muestra fecal. Además, en muestras de la misma paciente, fueron aisladas las especies Klebsiella pneumoniae resistente a carbapenemes por presencia de la misma enzima (KPC-2) en secreción orotraqueal y Enterococcus faecalis resistente a vancomicina en muestra fecal. Este hallazgo, confirma la portación del gen que codifica para la carbapenemasa tipo KPC-2 por cepa de Salmonella entérica, y que el mismo ocurre en un serotipo poco frecuente (serotipo Javiana). Además, la recuperación en otra muestra de la paciente de K. pneumoniae productora de carbapenemasa tipo KPC- 2 evidencia la diseminación de este mecanismo de resistencia entre las enterobacterias. Palabras claves: Salmonella, KPC, carbapenemasas, enterobacterias.

At the global level have emerged over the years, microorganisms resistant to various antimicrobial agents that have been incorporated to the therapeutic arsenal. Several mechanisms of resistance have emerged in recent years, highlighting the emergence of carbapenemases in enterobacterias, entity which Klebsiella pneumoniae carbapenemasa (KPC), being one of the most important, originally identified in the United States in 1996. In Paraguay, the first isolates of KPC carbapenemasecarrying strains were obtained in september 2009, from which the presence of several Enterobacteria species was confirmed, with Klebsiella pneumoniae being the most prevalent among them. According to data from the reference center, Salmonella enterica serotype Typhimurium is the most common serovar identified in stool samples to date, and the Javiana serotype the fourth. This report describes the first KPC-2 carbapenemase isolate in Salmonella enterica serovar Javiana isolated in February 2015 from a fecal sample. In addition, in samples from the same patient, Klebsiella pneumoniae species resistant to carbapenems were isolated by the presence of the same enzyme (KPC-2) in orotracheal secretion and vancomycin-resistant Enterococcus faecalis in fecal samples. Key words: Salmonella, KPC, carbapenemases, enterobacterias.

Recent Emergence of the Mild Strain of Tomato yellow leaf curl virus as a Cause of Tomato Yellow Leaf Curl Disease of Processing Tomatoes (Solanum lycopersicon) in the Dominican Republic.

In the early 1990s, the monopartite begomovirus Tomato yellow leaf curl virus (TYLCV) was introduced into the Dominican Republic (DO), and molecular characterization revealed it was an isolate of TYLCV-Israel (TYLCV-IL[DO]) (3,5). In 2006, a study of the variability of TYLCV in DO revealed that TYLCV-IL[DO] was associated with all samples of tomato yellow leaf curl (TYLC) tested and, thus, that the virus had been genetically stable for >15 years (2). However, in 2010 and 2011, 2 of 10 and 11 of 18 samples of TYLC, respectively, were negative for TYLCV infection based upon PCR with the TYLCV-specific primer pair, 2560v (5'-GAGAACAATTGGGATATG-3')/1480c (5'-AATCATGGATTCACGCAC-3'), which directs the amplification of a ~1.7 kb fragment. In 2011, two such samples from the Azua Valley were tested by PCR with the 1470v (5'-AGTGATGAGTTCCCCTGTGC-3')/UPC2 primer pair (1), and sequence analysis of the ~0.4 kb fragment amplified from both samples revealed infection with the mild strain of TYLCV (TYLCV-Mld). A primer specific for TYLCV-Mld was designed (2070v, 5'-AAACGGAGAAATATATAAGGAGCC-3'), and PCR with the 2070v/1480c primer pair directed the amplification of the expected ~2.1 kb fragment from all 11 TYLC samples collected in 2011 that were PCR-negative for TYLCV-IL[DO] infection. Sequence analyses confirmed these were TYLCV-Mld fragments. The complete TYLCV-Mld genome was amplified from two samples from the Azua Valley with Templiphi, the amplified DNA products digested with Sal I, and the resulting ~2.8 kb fragments ligated into Sal I-digested pGEM-11. The complete sequences of these isolates were 2,791 nt and 99% identical to each other and 98% identical to sequences of TYLCV-Mld isolates. The TYLCV-Mld isolates from the DO were designated TYLCV-Mld:DO:TY5:01:2011 (KJ913682) and TYLCV-Mld:DO:TY5:02:2011 (KJ913683). A multimeric clone of TYLCV-Mld:DO:TY5:01:2011 was generated in the binary vector pCAMBIA1300 by cloning a 2.2 kb Sal I-EcoRI fragment containing the intergenic region to generate a 0.8-mer (pCTYMld0.8), and then the full-length Sal I fragment was cloned into the Sal I site of pCTYMld0.8 to generate a 1.8-mer (pCTYMldDO-01-1.8). Tomato plants agroinoculated with Agrobacterium tumefaciens carrying pCTYMldDO-01-1.8 developed severe TYLC disease symptoms 10 to 14 days after inoculation, whereas plants inoculated with a strain carrying the empty vector did not develop symptoms. Samples of processing tomatoes with TYLC were collected in 2012 to 2014 in the DO and tested for TYLCV-IL[DO] and TYLCV-Mld by PCR with the 2560v/1480c and 2070v/1480c primers pairs, respectively; these samples had infections of 93% (13/14), 86% (18/21), and 61% (11/18) with TYLCV-Mld; 29% (4/14), 19% (4/21), and 56% (10/18) with TYLCV-IL[DO]; and 21% (3/14), 5% (1/21), and 28% (5/18) with both viruses, respectively. These results reveal that there has been a striking population shift in the begomovirus causing TYLC in the DO, with TYLCV-Mld becoming predominant. This may reflect selection pressure(s) favoring a small pre-existing population of TYLCV-Mld, such as new tomato varieties, or a recent introduction event, such as that described in Venezuela (4). References: (1) R. W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (2) R. L. Gilbertson et al. Page 279 in: Tomato yellow leaf curl virus disease. Springer, 2007. (3) M. K. Nahkla et al. Plant Dis. 78:926, 1994. (4) G. Romay et al. Australasian Plant Dis. Notes, in press, 2014. (5) R. Salati et al. Phytopathology 92:487, 2002.

Natural History of Innate Host Defense Peptides.

Host defense peptides act on the forefront of innate immunity, thus playing a central role in the survival of animals and plants. Despite vast morphological changes in species through evolutionary history, all animals examined to date share common features in their innate immune defense strategies, hereunder expression of host defense peptides (HDPs). Most studies on HDPs have focused on humans, domestic and laboratory animals. More than a thousand different sequences have been identified, yet data on HDPs in wild-living animals are sparse. The biological functions of HDPs include broad-spectrum antimicrobial activity and immunomodulation. Natural selection and coevolutionary host-pathogen arms race theory suggest that the extent and specificity of the microbial load influences the spectrum and potency of HDPs in different species. Individuals of extant species-that have lived for an extended period in evolutionary history amid populations with intact processes of natural selection-likely possess the most powerful and well-adapted "natural antibiotics". Research on the evolutionary history of the innate defense system and the host in context of the consequences of challenges as well as the efficacy of the innate immune system under natural conditions is therefore of immediate interest. This review focuses on evolutionary aspects of immunophysiology, with emphasis on innate effector molecules. Studies on host defense in wild-living animals may significantly enhance our understanding of inborn immune mechanisms, and help identify molecules that may assist us to cope better with the increasing microbial challenges that likely follow from the continuous amplification of biodiversity levels on Earth.

Innate immunity and host defense peptides in veterinary medicine.

Recent years have witnessed a surge in interest directed at innate immune mechanisms. Proper conceptualization of the key elements of innate immunity, however, is still a work in progress, because most research in immunology traditionally has been focused on components of the acquired immune response. The question of why an animal stays healthy in a world filled with many dangers is perhaps as interesting as why it sometimes surrenders to disease. Consequently, studies with an increased focus on inborn mechanisms of animal host defense may help further the development of appropriate preventative and therapeutic measures in veterinary medicine. Host defense peptides (HDPs) are central effector molecules of innate immunity, and are produced by virtually all living species throughout the plant and animal kingdoms. These gene-encoded peptides play a central role in multiple, clinically relevant disease processes. Imbalances in the expression of HDPs can lead to overt pathology in different organ systems and cell types in all species studied. In addition, HDPs are an ancient group of innate chemical protectors, which are now evaluated as model molecules for the development of novel natural antibiotics and immunoregulatory compounds. This review provides an overview of HDPs and is aimed at veterinary practitioners as well as basic researchers with an interest in comparative immunology involving small and large animal species.

Strains of BCMV and BCMNV characterized from lima bean plants affected by deforming mosaic disease in Peru.

Viruses of the species Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) were simultaneously detected by the different size of PCR amplicons in lima bean plants (Phaseolus lunatus) displaying deforming mosaic symptoms in Peru. Phylogenetic analysis of partial deduced CP amino acid sequences indicated that the Peruvian BCMV isolates belong to new strains. One isolate differed from the other Peruvian isolates, and also from the ten previously described BCMV strains recognized by responses on differential bean varieties. The sequence of the 3'-proximal part (2547 nucleotides) of the genome confirmed that this isolate also belongs to BCMV.

Strains of Peru tomato virus infecting cocona (Solanum sessiliflorum), tomato and pepper in Peru with reference to genome evolution in genus Potyvirus.

Two isolates (SL1 and SL6) of Peru tomato virus (PTV, genus Potyvirus) were obtained from cocona plants (Solanum sessiliflorum) growing in Tingo María, the jungle of the Amazon basin in Peru. One PTV isolate (TM) was isolated from a tomato plant (Lycopersicon esculentum) growing in Huaral at the Peruvian coast. The three PTV isolates were readily transmissible by Myzus persicae. Isolate SL1, but not SL6, caused chlorotic lesions in inoculated leaves of Chenopodium amaranticolor and C. quinoa. Isolate TM differed from SL1 and SL6 in causing more severe mosaic symptoms in tomato, and vein necrosis in the leaves of cocona. Pepper cv. Avelar (Capsicum annuum) showed resistance to the PTV isolates SL1 and SL6 but not TM. The 5'- and 3'-proximal sequences of the three PTV isolates were cloned, sequenced and compared to the corresponding sequences of four PTV isolates from pepper, the only host from which PTV isolates have been previously characterised at the molecular level. Phylogenetic analyses on the P1 protein and coat protein amino acid sequences indicated, in accordance with the phenotypic data from indicator hosts, that the PTV isolates from cocona represented a distinguishable strain. In contrast, the PTV isolates from tomato and pepper were not grouped according to the host. Inclusion of the sequence data from the three PTV isolates of this study in a phylogenetic analysis with other PTV isolates and other potyviruses strengthen the membership of PTV in the so-called "PVY subgroup" of Potyvirus. This subgroup of closely related potyvirus species was also distinguishable from other potyviruses by their more uniform sizes of the protein-encoding regions within the polyprotein.

Comparison of tear proteins of llamas and cattle.

OBJECTIVE: To analyze and compare contents of the preocular tear films of llamas and cattle. ANIMALS: 40 llamas and 35 cattle. PROCEDURE: Tear pH was determined by use of a pH meter. Total protein concentration was determined by use of 2 microtiter methods. Tear proteins were separated by use of electrophoresis and molecular weights of bands were calculated. Western blot immunoassay was used to detect IgA, lactoferrin, transferrin, ceruloplasmin, alpha1-antitrypsin, alpha1-amylase, and alpha2-macroglobulin. Enzyme electrophoresis was used to detect proteases. RESULTS: The pH of llama and cattle tears were 8.05 +/- 0.01 and 8.10 +/- 0.01, respectively. For results of both methods, total protein concentration of llama tears was significantly greater than that of cattle tears. Molecular weights of tear protein bands were similar within and between the 2 species, although llama tears had a distinct 13.6-kd band that was not detected in cattle. Lactoferrin, IgA, transferrin, ceruloplasmin, alpha1-antitrypsin, alpha1-amylase, alpha2-macroglobulin, and proteases were detected in both species. CONCLUSIONS AND CLINICAL RELEVANCE: Llama tears have significantly greater total protein concentration than cattle tears, whereas pH is similar between species. Because little variation was detected within species for the number and molecular weight of protein bands, pooling of tears for analysis is justified. Results suggest that lactoferrin, ceruloplasmin, transferrin, alpha1-antitrypsin, alpha2-macroglobulin, alpha1-amylase, and IgA are present in the tears of llamas and cattle.

Development and validation of an enzyme-linked immunosorbent assay for feline trypsin-like immunoreactivity.

OBJECTIVE: To develop and validate an ELISA for quantitative analysis of feline trypsin-like immunore-activity (fTLI). SAMPLE POPULATION: Purified feline cationic trypsin (fCT) and rabbit anti-fCT antiserum; blood samples from 63 healthy cats. PROCEDURES: A sandwich capture ELISA was developed, using anti-fCT antiserum purified by affinity chromatography that underwent biotinylation. Purified fCT was used for standards. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. A reference range was established by assaying serum samples from the 63 healthy cats. RESULTS: Sensitivity was 1.23 microg/L; working range was 2 to 567 microg/L. Ratios of observed versus expected results for 4 samples tested at various dilutions ranged from 90.0 to 120.7%. Ratios of observed versus expected results for 5 samples spiked with various concentrations of fCT ranged from 82.0 to 101.8%. Intra- and inter-assay coefficients of variability ranged from 9.9 to 11.1% and from 10.2 to 21.7%, respectively. The reference range for serum fTLI measured with this ELISA was 12 to 82 microg/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that an ELISA can be used to measure serum fTLI in cats. The ELISA was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use.

Serum unconjugated bile acids as a test for intestinal bacterial overgrowth in dogs.

Small intestinal bacterial overgrowth (SIBO) has a high incidence in dogs and, as in humans, is difficult to diagnose. The aim of this study was to determine the diagnostic significance of serum unconjugated bile acid concentrations in dogs with bacterial overgrowth. Fasting sera were obtained from 23 dogs: 10 with culture-proven SIBO, 8 with indirectly diagnosed SIBO (normal pancreatic function but small intestinal disease associated with subnormal serum cobalamin and supranormal folate concentrations), and 5 healthy controls. Unconjugated bile acids were determined using gas chromatography-mass spectrometry after isolation by liquid-solid extraction and anion-exchange chromatography. Mean serum unconjugated bile acid concentrations were significantly elevated in dogs with SIBO (mean +/- SD: 0.91 +/- 1.03 micromol/liter), and in dogs with indirectly diagnosed SIBO (2.11 +/- 2.20 micromol/liter) compared to clinically healthy dogs (0.015 +/- 0.015 micromol/liter, P < 0.005). Cholic acid was the predominant unconjugated bile acid in the serum of dogs with SIBO. In conclusion serum unconjugated bile acid concentrations of healthy dogs are significantly lower than reported values for humans, and this fraction represents a relatively small proportion (0-2.3%; mean 0.8%) of the total bile acids in dogs. Unconjugated bile acids increased 10- to 20-fold in dogs with SIBO indicating the clinical utility of serum unconjugated bile acids for diagnosis of intestinal bacterial overgrowth in dogs.

Enzyme-linked immunosorbent assay for canine alpha 1-protease inhibitor.

OBJECTIVE: To develop and validate an ELISA for quantifying alpha 1-protease inhibitor (alpha 1-PI) in serum and fecal extracts. PROCEDURE: Affinity-purified rabbit origin canine alpha 1-PI antibodies were biotinylated and, after addition of streptavidin-horseradish peroxidase, were used as the labeled complex in a noncompetitive immunoassay. The alpha 1-PI standards were made from purified serum canine alpha 1-PI diluted in phosphate-buffered saline solution containing 5% newborn calf serum and 0.01% thimerosal. This assay was validated by determining linearity, recovery of added alpha 1-PI, detection limit, and intra- and interassay precision. Control range for serum and fecal alpha 1-PI concentration was determined for samples from 25 healthy pet dogs. RESULTS: The standard curve was linear between 5 and 50 ng/ml. Curves plotted after serial dilution of serum also were linear and ran parallel to the standard curve. Between- and within-assay coefficients of variation were 9.1 and 8.7%, respectively. The accuracy of the assay was tested by measuring the recovery of alpha 1-PI added to serum and fecal extracts and ranged from 93 to 111%. The reference range of fecal alpha 1-PI concentration was 0.023 to 5.67 (mean +/- SD, 2.0 +/- 1.82) micrograms/g and of serum alpha 1-PI was 0.901 to 1.96 (1.42 +/- 0.32) g/L. CONCLUSIONS: This ELISA had high precision, accuracy, and reproducibility for quantification of canine alpha 1-PI concentration in serum and fecal extracts. CLINICAL RELEVANCE: Specific ELISA for alpha 1-PI may be a useful test for the diagnosis of protein-losing enteropathy in dogs, and as such, will facilitate further characterization and better understanding of this canine disorder.

Isolation and characterization of alpha 1-protease inhibitor from canine plasma.

OBJECTIVE: To improve a previously described purification process by producing a higher yield and purity of alpha 1-protease inhibitor (alpha 1-PI) from canine plasma. ANIMALS: Plasma pool from 10 clinically normal male dogs. PROCEDURE: Canine alpha 1-PI was purified by use of ammonium sulfate precipitation, ion-exchange chromatography, and 3 affinity chromatographic procedures: concanavalin A-Sepharose, thiol, and hemoglobin-Sepharose. Characterization was performed by gel electrophoresis, isoelectric focusing, and immunoblot analysis. The N-terminal amino acid sequence was obtained by use of the Edman degradation method and a gas amino acid sequencer. RESULTS: Canine alpha 1-PI was purified with a yield of approximately 7% and a 54-fold increase in specific inhibitory activity. The inhibitor had a molecular weight of 59,000 and had 2 major patterns after isoelectric focusing: fast and intermediate in homozygous and/or heterozygous forms. Edman degradation revealed glutamic acid as the starting amino acid from the N-terminal sequence. Homologies of the N-terminal sequence of canine alpha 1-PI with those of sheep, horse, and human alpha 1-protease inhibitors were 54, 46, and 41%, respectively. CONCLUSIONS: Canine protease inhibitor is analogous to the alpha 1-protease inhibitors of sheep, human beings, and mice in terms of molecular weight, amino acid composition, and inhibitory activity against trypsin. Although the method described had a yield of 7%, the final product retained inhibitory activity and was pure. CLINICAL RELEVANCE: The availability of pure canine alpha 1-PI, as well as the specific antibodies, will facilitate studies on the fecal excretion and structural heterogeneity of this protein in dogs with naturally acquired protein-losing enteropathy.

Diagnostico y mmanejo del carcinoma inflamatorio de la glandula mamaria / Diagnosis and management of inflamatory breast cancer

Debido al funesto resultado de la mastectomia el rol de la cirugia se limito a la biopsia y accedio a la terapia por radiacion en forma primaria. Este tratamiento dio como resultado un mejor control local para la reduccion de la incidencia de la ulceracion cutanea, pero no mejoro la sobre vida. Los indices muestran una sobre vida de 4 a 6 meses en la mayoria de los pacientes. Este tratamiento cambinado dio una alta incidencia de recurrencia local y proporciono una sobrevida media de 18 meses