Serovars, Virulence and Antimicrobial Resistance Genes of Non-Typhoidal Salmonella Strains from Dairy Systems in Mexico.

Salmonella isolated from dairy farms has a significant effect on animal health and productivity. Different serogroups of Salmonella affect both human and bovine cattle causing illness in both reservoirs. Dairy cows and calves can be silent Salmonella shedders, increasing the possibility of dispensing Salmonella within the farm. The aim of this study was to determine the genomic characteristics of Salmonella isolates from dairy farms and to detect the presence of virulence and antimicrobial resistance genes. A total of 377 samples were collected in a cross-sectional study from calves, periparturient cow feces, and maternity beds in 55 dairy farms from the states of Aguascalientes, Baja California, Chihuahua, Coahuila, Durango, Mexico, Guanajuato, Hidalgo, Jalisco, Queretaro, San Luis Potosi, Tlaxcala, and Zacatecas. Twenty Salmonella isolates were selected as representative strains for whole genome sequencing. The serological classification of the strains was able to assign groups to only 12 isolates, but with only 5 of those being consistent with the genomic serotyping. The most prevalent serovar was Salmonella Montevideo followed by Salmonella Meleagridis. All isolates presented the chromosomal aac(6')-Iaa gene that confers resistance to aminoglycosides. The antibiotic resistance genes qnrB19, qnrA1, sul2, aph(6)-Id, aph(3)-ld, dfrA1, tetA, tetC, flor2, sul1_15, mph(A), aadA2, blaCARB, and qacE were identified. Ten pathogenicity islands were identified, and the most prevalent plasmid was Col(pHAD28). The main source of Salmonella enterica is the maternity areas, where periparturient shedders are contaminants and perpetuate the pathogen within the dairy in manure, sand, and concrete surfaces. This study demonstrated the necessity of implementing One Health control actions to diminish the prevalence of antimicrobial resistant and virulent pathogens including Salmonella.

High Frequency of Antibiotic Resistance Genes (ARGs) in the Lerma River Basin, Mexico.

The spread of beta-lactamase-producing bacteria is of great concern and the environment has been found to be a main source of contamination. Herein, it was proposed to determine the frequency of antimicrobial-resistant-Gram-negative bacteria throughout the Lerma River basin using phenotypic and molecular methods. Resistant bacteria were isolated with chromogenic media and antimicrobial susceptibility tests were used to characterize their resistance. ARGs for beta-lactams, aminoglycosides, and quinolones were detected by PCR. Species were identified by Sanger sequencing the 16S rRNA gene and the representative genomes of MDR strains were sequenced by NGS. A high variation in the number of isolates was observed in the 20 sampled sites, while observing a low diversity among the resistant bacteria. Of the 12 identified bacterial groups, C. freundii, E. coli, and S. marcescens were more predominant. A high frequency of resistance to beta-lactams, quinolones, and aminoglycosides was evidenced, where the blaCTX,qnrB, qnrS y, and aac(6')lb-cr genes were the most prevalent. C. freundii showed the highest frequency of MDR strains. Whole genome sequencing revealed that S. marcescens and K. pneumoniae showed a high number of shared virulence and antimicrobial resistance genes, while E. coli showed the highest number of unique genes. The contamination of the Lerma River with MDR strains carrying various ARGs should raise awareness among environmental authorities to assess the risks and regulations regarding the optimal hygienic and sanitary conditions for this important river that supports economic activities in the different communities in Mexico.

Characteristics of dual carbapenemase-producing Klebsiella pneumoniae strains from an outbreak in Venezuela: a retrospective study.

OBJECTIVE: To characterize carbapenemase-producing Klebsiella pneumoniae isolated from patients treated at a hospital in Cumaná, Sucre, Venezuela. METHODS: This was a retrospective study conducted at the general hospital in Cumaná where 58 K. pneumoniae strains were analyzed for resistance to antimicrobials, specifically carbapenems, in January - June 2015. Production of metallo-ß-lactamases and serine carbapenemases was determined by the double-disc synergy test, using EDTA-sodium mercaptoacetic acid and 3-aminophenyl boronic acid discs, respectively. Multiplex-PCR was used to detect genes coding for carbapenemases. Molecular typing using ERIC-PCR determined the presence of clones. RESULTS: Four strains of K. pneumoniae resistant to carbapenems were identified. Phenotypic methods for detection of metallo-ß-lactamases and serine carbapenemases were positive, and PCR demonstrated the co-presence of bla NDM and bla KPC genes in all four strains. ERIC-PCR identified two clones circulating in the hospital. CONCLUSIONS: Infection control strategies are needed at the central hospital in Cumaná and its surrounding areas to prevent the spread of these pathogens, especially given the high levels of migration from Venezuela to other countries in South America.

New Perspectives Related to the Bioluminescent System in Dinoflagellates: Pyrocystis lunula, a Case Study.

Pyrocystis lunula is considered a model organism due to its bioluminescence capacity linked to circadian rhythms. The mechanisms underlying the bioluminescent phenomenon have been well characterized in dinoflagellates; however, there are still some aspects that remain an enigma. Such is the case of the presence and diversity of the luciferin-binding protein (LBP), as well as the synthesis process of luciferin. Here we carry out a review of the literature in relation to the molecular players responsible for bioluminescence in dinoflagellates, with particular interest in P. lunula. We also carried out a phylogenetic analysis of the conservation of protein sequence, structure and evolutionary pattern of these key players. The basic structure of the luciferase (LCF) is quite conserved among the sequences reported to date for dinoflagellate species, but not in the case of the LBP, which has proven to be more variable in terms of sequence and structure. In the case of luciferin, its synthesis has been shown to be complex process with more than one metabolic pathway involved. The glutathione S-transferase (GST) and the P630 or blue compound, seem to be involved in this process. In the same way, various hypotheses regarding the role of bioluminescence in dinoflagellates are exposed.

Characteristics of dual carbapenemase-producing Klebsiella pneumoniae strains from an outbreak in Venezuela: a retrospective study

[ABSTRACT]. Objective. To characterize carbapenemase-producing Klebsiella pneumoniae isolated from patients treated at a hospital in Cumaná, Sucre, Venezuela. Methods. This was a retrospective study conducted at the general hospital in Cumaná where 58 K. pneumoniae strains were analyzed for resistance to antimicrobials, specifically carbapenems, in January – June 2015. Production of metallo-β-lactamases and serine carbapenemases was determined by the double-disc synergy test, using EDTA-sodium mercaptoacetic acid and 3-aminophenyl boronic acid discs, respectively. Multiplex-PCR was used to detect genes coding for carbapenemases. Molecular typing using ERIC-PCR determined the presence of clones. Results. Four strains of K. pneumoniae resistant to carbapenems were identified. Phenotypic methods for detection of metallo-β-lactamases and serine carbapenemases were positive, and PCR demonstrated the co-presence of blaNDM and blaKPC genes in all four strains. ERIC-PCR identified two clones circulating in the hospital. Conclusions. Infection control strategies are needed at the central hospital in Cumaná and its surrounding areas to prevent the spread of these pathogens, especially given the high levels of migration from Venezuela to other countries in South America.

[RESUMEN]. Objetivo. Caracterizar la Klebsiella pneumoniae productora de carbapenemasa aislada de pacientes tratados en un hospital de Cumaná (Sucre, Venezuela). Métodos. Se hizo un estudio retrospectivo en el hospital central de Cumaná, donde se analizaron 58 cepas de k. pneumoniae para estudiar la resistencia a los antimicrobianos, específicamente a los fármacos carbapenémicos, entre enero y junio del 2015. La producción de metalo-β-lactamasas y carbapenemasas de serina se determinó mediante la prueba de sinergia de doble disco, usando discos de EDTA SMA de sodio y de ácido borónico 3 aminofenil, respectivamente. Se usó la PCR múltiple para detectar la codificación de genes correspondiente a las carbapenemasas. Se determinó la presencia de clones por tipificación molecular mediante la técnica de ERIC PCR. Resultados. Se detectaron cuatro cepas de K. pneumoniae resistentes a los fármacos carbapenémicos. Los métodos fenotípicos para la detección de metalo-β-lactamasas y carbapenemasas de serina fueron positivos y se demostró mediante la PCR la copresencia de los genes blaNDM y blaKPC en las cuatro cepas. Por medio de la técnica ERIC-PCR se detectaron dos clones que circulaban en el hospital. Conclusiones. Es necesario adoptar estrategias de control de infecciones en el hospital central en Cumaná y las zonas circundantes para prevenir la propagación de estos agentes patógenos, especialmente dados los niveles altos de migración de Venezuela a otros países de América del Sur.

[RESUMO]. Objetivo. Caracterizar cepas de Klebsiella pneumoniae produtoras de carbapenemases isoladas de pacientes tratados em um hospital em Cumaná, Sucre, na Venezuela. Métodos. Realizamos um estudo retrospectivo no hospital geral de Cumaná, onde 58 cepas de K. pneumoniae foram analisadas para verificar a resistência a antimicrobianos, especificamente carbapenens, entre janeiro e junho de 2015. A produção de metalo-β-lactamases e serino-carbapenemases foi determinada pelo teste de sinergia de disco duplo, usando discos de EDTA sódico-ácido mercaptoacético e ácido 3-aminofenil borônico, respectivamente. Utilizamos a PCR multiplex para detectar os genes codificadores de carbapenemases. A tipagem molecular por ERIC-PCR determinou a presença de clones. Resultados. Foram identificadas quatro cepas de K. pneumoniae resistentes a carbapenens. Os métodos fenotípicos para a detecção de metalo-β-lactamases e serino-carbapenemases foram positivos, e a PCR demonstrou a co-presença dos genes blaNDM e blaKPC em todas as quatro cepas. A ERIC-PCR identificou dois clones que circulavam no hospital. Conclusões. São necessárias estratégias de controle de infecções no hospital central de Cumaná e seus arredores para prevenir a disseminação destes patógenos, especialmente devido aos altos níveis de migração da Venezuela para outros países da América do Sul.

Multidrug resistance and risk factors associated with community-acquired urinary tract infections caused by Escherichia coli in Venezuela. / Multirresistencia a medicamentos y factores de riesgo asociados con infecciones urinarias por Escherichia coli adquiridas en la comunidad, Venezuela

INTRODUCTION: The treatment of urinary tract infections has become more challenging due to the increasing frequency of multidrug-resistant Escherichia coli in human populations. OBJECTIVE: To characterize multidrug-resistant E. coli isolates causing community-acquired urinary tract infections in Cumaná, Venezuela, and associate possible risk factors for infection by extended-spectrum beta-lactamases (ESBL)-producing isolates. MATERIALS AND METHODS: We included all the patients with urinary tract infections attending the urology outpatient consultation and emergency unit in the Hospital de Cumaná, Estado Sucre, Venezuela, from January through June, 2014. blaTEM, blaSHV and blaCTX-M genes detection was carried out by PCR. RESULTS: We found a high prevalence of multidrug-resistant E. coli (25.2%) with 20.4% of the isolates producing ESBL. The ESBL-producing isolates showed a high frequency (66.7%) of simultaneous resistance to trimethoprim-sulphamethoxazole, fluoroquinolones and aminoglycosides compared to non-producing isolates (2.4%). Of the resistant isolates, 65.4% carried the blaTEM gene, 34.6% the blaCTX-M and 23.1% the blaSHV. The blaCTX-M genes detected belonged to the CTX-M-1 and CTX-M-2 groups. Plasmid transfer was demonstrated by in vitro conjugation in 17 of the 26 ESBL-producing isolates. All three genes detected were transferred to the transconjugants. Age over 60 years, complicated urinary tract infections and previous use of a catheter predisposed patients to infection by ESBL-producing E. coli. CONCLUSIONS: The high frequency of multidrug-resistant ESBL-producing isolates should alert the regional health authorities to take measures to reduce the risk of outbreaks caused by these types of bacteria in the community.

Introducción. El tratamiento de las infecciones urinarias constituye un reto creciente por el aumento de Escherichia coli proveniente de la comunidad multirresistente a los medicamentos. Objetivo. Caracterizar aislamientos de E. coli multirresistente causantes de infecciones urinarias adquiridas en la comunidad en Cumaná, Venezuela, y detectar los posibles riesgos de infección por aislamientos productores de betalactamasas de espectro extendido (BLEE). Materiales y métodos. Se incluyeron todos los pacientes atendidos en la consulta externa de urología y en urgencias del Hospital de Cumaná entre enero y junio de 2014 y que evidenciaban infecciones urinarias. La detección de los genes blaTEM, blaSHV y blaCTX-M se hizo mediante la reacción en cadena de la polimerasa (PCR). Resultados. Se encontró una alta prevalencia de E. coli multirresistente a los medicamentos (25,2 %), con 20,4 % de aislamientos productores de BLEE y una gran frecuencia de resistencia simultánea a trimetoprim-sulfametoxazol, fluoroquinolonas y aminoglucósidos (66,7 %) comparados con los no productores (2,4 %). En el 65,4 % de los aislamientos resistentes, se encontró el gen blaTEM; en 34,6 %, el blaCTX-M, y en 23,1 %, el blaSHV. Los genes blaCTX-M detectados pertenecían a los grupos CTX-M-1 y CTX-M-2. Se demostró la transferencia in vitro de plásmidos por conjugación en 17 de los 26 aislamientos productores de BLEE. Los tres tipos de genes detectados se transfirieron a los transconjugantes. La edad mayor de 60 años, las infecciones urinarias con complicaciones y el uso previo de catéter, predispusieron a la infección por cepas de E. coli productoras de BLEE. Conclusiones. La gran frecuencia de aislamientos multirresistentes productores de BLEE debería alertar a las autoridades sanitarias para tomar medidas que reduzcan el riesgo de epidemias causadas por este tipo de bacterias en la comunidad.

Multidrug resistance and risk factors associated with community-acquired urinary tract infections caused by Escherichia coli in Venezuela / Multirresistencia a medicamentos y factores de riesgo asociados con infecciones urinarias por Escherichia coli adquiridas en la comunidad, Venezuela

Abstract Introduction: The treatment of urinary tract infections has become more challenging due to the increasing frequency of multidrug-resistant Escherichia coli in human populations. Objective: To characterize multidrug-resistant E. coli isolates causing community-acquired urinary tract infections in Cumaná, Venezuela, and associate possible risk factors for infection by extended-spectrum beta-lactamases (ESBL)-producing isolates. Materials and methods: We included all the patients with urinary tract infections attending the urology outpatient consultation and emergency unit in the Hospital de Cumaná, Estado Sucre, Venezuela, from January through June, 2014. blaTEM, blaSHV and blaCTX-M genes detection was carried out by PCR. Results: We found a high prevalence of multidrug-resistant E. coli (25.2%) with 20.4% of the isolates producing ESBL. The ESBL-producing isolates showed a high frequency (66.7%) of simultaneous resistance to trimethoprim-sulphamethoxazole, fluoroquinolones and aminoglycosides compared to non-producing isolates (2.4%). Of the resistant isolates, 65.4% carried the blaTEM gene, 34.6% the blaCTX-M and 23.1% the blaSHV. The blaCTX-M genes detected belonged to the CTX-M-1 and CTX-M-2 groups. Plasmid transfer was demonstrated by in vitro conjugation in 17 of the 26 ESBL-producing isolates. All three genes detected were transferred to the transconjugants. Age over 60 years, complicated urinary tract infections and previous use of a catheter predisposed patients to infection by ESBL-producing E. coli. Conclusions: The high frequency of multidrug-resistant ESBL-producing isolates should alert the regional health authorities to take measures to reduce the risk of outbreaks caused by these types of bacteria in the community.

Resumen Introducción. El tratamiento de las infecciones urinarias constituye un reto creciente por el aumento de Escherichia coli proveniente de la comunidad multirresistente a los medicamentos. Objetivo. Caracterizar aislamientos de E. coli multirresistente causantes de infecciones urinarias adquiridas en la comunidad en Cumaná, Venezuela, y detectar los posibles riesgos de infección por aislamientos productores de betalactamasas de espectro extendido (BLEE). Materiales y métodos. Se incluyeron todos los pacientes atendidos en la consulta externa de urología y en urgencias del Hospital de Cumaná entre enero y junio de 2014 y que evidenciaban infecciones urinarias. La detección de los genes blaTEM, blaSHV y blaCTX-M se hizo mediante la reacción en cadena de la polimerasa (PCR). Resultados. Se encontró una alta prevalencia de E. coli multirresistente a los medicamentos (25,2 %), con 20,4 % de aislamientos productores de BLEE y una gran frecuencia de resistencia simultánea a trimetoprim-sulfametoxazol, fluoroquinolonas y aminoglucósidos (66,7 %) comparados con los no productores (2,4 %). En el 65,4 % de los aislamientos resistentes, se encontró el gen blaTEM; en 34,6 %, el blaCTX-M, y en 23,1 %, el blaSHV. Los genes blaCTX-M detectados pertenecían a los grupos CTX-M-1 y CTX-M-2. Se demostró la transferencia in vitro de plásmidos por conjugación en 17 de los 26 aislamientos productores de BLEE. Los tres tipos de genes detectados se transfirieron a los transconjugantes. La edad mayor de 60 años, las infecciones urinarias con complicaciones y el uso previo de catéter, predispusieron a la infección por cepas de E. coli productoras de BLEE. Conclusiones. La gran frecuencia de aislamientos multirresistentes productores de BLEE debería alertar a las autoridades sanitarias para tomar medidas que reduzcan el riesgo de epidemias causadas por este tipo de bacterias en la comunidad.

Virulence factors and integrons are associated with MDR and XDR phenotypes in nosocomial strains of Pseudomonas aeruginosa in a Venezuelan university hospital.

Multidrug resistance (MDR), virulence and transferable elements potentiate Pseudomonas aeruginosa's role as an opportunistic pathogen creating a high risk for public health. In this study, we evaluated the possible association of multidrug resistance, virulence factors and integrons with intrahospital P. aeruginosa strains isolated from patients at Cumana hospital, Venezuela. Relevant clinical-epidemiological data were collected to study 176 strains (2009-2016) isolated from different hospital units. Bacterial resistance was classified as susceptible, low-level resistant (LDR), multidrug resistant (MDR) and extensively drug-resistant (XDR). Most strains produced pyoverdine, DNase, gelatinase and hemolysin. Around 73% of the strains showed some type of movement. MDR and XDR strains increased from 2009 (24.2% and 4.8%, respectively) to 2016 (53.1% and 18.8%); while LDR decreased from 64.5% to 6.3%. The exoU and exoS genes were found in a significant number of strains (38.1 and 7.4%, respectively). Class I integrons were detected in 35.8% of the strains and the frequency was associated with resistance (42.9, 22.4, 41.4 and 61.9%, for susceptible, LDR, MDR and XDR, respectively). The MDR/XDR strains were positively associated with hemolysins and exoU, but negatively associated with bacterial twitching. MDR/XDR phenotypes were also associated with the Intensive Care Unit (ICU), septicemia, bronchial infection and diabetic foot ulcers, as well as long hospital stay (≥10 days) and previous antimicrobial treatment. High frequency of MDR/XDR strains and their association with class I integrons and virulence factors can increase the infection potential, as well as morbidity and mortality of patients attending this hospital and could spread infection to the community, creating a health risk for the region.

[qnr genes in Enterobacteriaceae isolated from at a hospital in Venezuela]. / Genes qnr en Enterobacteriaceae aisladas en un hospital de Venezuela.

BACKGROUND: Enterobacteria resistant to quinolones is increasing worldwide, including Venezuela. The mechanism for this resistance could be due to genes included in the chromosome or in transmissible plasmids. AIM: To evaluate the resistance to quinolones, coded by qnr genes present in enterobacteria species, isolated in the University Hospital of Cumana, Venezuela. METHODS: Antimicrobial susceptibility tests to quinolones, beta-lactams and aminoglycosides were carried out to all the isolates. The presence of qnr genes were determined by PCR. The isolates carrying the qnr genes were used for bacterial conjugation tests to determine the presence of transferable plasmids. Antimicrobial susceptibility tests and PCR were carried out in the transconjugants to verify the transfer of the genes. RESULTS: High levels of antimicrobial resistance to quinolones and beta-lactams were found among the isolates. We found that 33.6% of the isolates carry the qnrB gene and 0.9% qnr A gene. Of the 23 transconjugants, 20 showed to have qnrB gene, but none qnrA. DISCUSSION: We concluded that the high frequency of qnr genes found in the enterobacteria isolates and their presence on transferable plasmids, complicate the use of quinolones for the treatment of bacterial infections, thus, a treatment plan should be designed with the rational use and the rotation of different types of antimicrobials, in order to avoid the selection of increasingly resistant strains.

Genes qnr en Enterobacteriaceae aisladas en un hospital de Venezuela / qnr genes in Enterobacteriaceae isolated from at a hospital in Venezuela

Resumen Introducción: La resistencia de enterobacterias a quinolonas se ha difundido por el mundo, fenómeno presente también en Venezuela. El mecanismo de esta resistencia pudiera estar mediado por genes incluidos en el cromosoma bacteriano o transmitirse en el interior de plásmidos. Objetivo: Evaluar la resistencia a quino-lonas, codificada por genes qnr, presentes en cepas de enterobacterias, aisladas en el Hospital Universitario de Cumaná, Venezuela. Métodos: A las cepas obtenidas se les realizaron pruebas de susceptibilidad antimicrobiana a quinolonas, β-lactámicos y aminoglucósidos. La presencia del gen qnr se determinó por RPC. Las enterobacterias portadoras del gen qnr fueron sometidas al proceso de conjugación bacteriana para comprobar su capacidad de transferencia. A las transconjugantes obtenidas se les realizó pruebas de susceptibilidad antimicrobiana y RPC para comprobar la transferencia de los genes. Resultados: Se encontraron elevados porcentajes de resistencia antimicrobiana a quinolonas y betalactámicos. El 33,6% de las cepas eran portadoras del gen qnrB, y 0,9% del gen qnrA. Se obtuvieron 23 cepas transconjugantes; de éstas, 20 portaban el gen qnrB, no se observó la presencia de qnrA. Discusión: En conclusión, el elevado porcentaje de genes qnr encontrado en las enterobacterias aisladas, y comprobada la presencia de éstos en plásmidos transferibles, complica la aplicación de tratamientos basados en quinolonas y fluoroquinolonas, por lo que es recomendable el uso racional de estos antimicrobianos, y proponer la rotación de la terapia antimicrobiana, a fin de evitar la selección de cepas resistentes.

Background: Enterobacteria resistant to quinolones is increasing worldwide, including Venezuela. The mechanism for this resistance could be due to genes included in the chromosome or in transmissible plasmids. Aim: To evaluate the resistance to quinolones, coded by qnr genes present in enterobacteria species, isolated in the University Hospital of Cumana, Venezuela. Methods: Antimicrobial susceptibility tests to quinolones, beta-lactams and aminoglycosides were carried out to all the isolates. The presence of qnr genes were determined by PCR. The isolates carrying the qnr genes were used for bacterial conjugation tests to determine the presence of transferable plasmids. Antimicrobial susceptibility tests and PCR were carried out in the transconjugants to verify the transfer of the genes. Results: High levels of antimicrobial resistance to quinolones and beta-lactams were found among the isolates. We found that 33.6% of the isolates carry the qnrB gene and 0.9% qnr A gene. Of the 23 transconjugants, 20 showed to have qnrB gene, but none qnrA. Discussion: We concluded that the high frequency of qnr genes found in the enterobacteria isolates and their presence on transferable plasmids, complicate the use of quinolones for the treatment of bacterial infections, thus, a treatment plan should be designed with the rational use and the rotation of different types of antimicrobials, in order to avoid the selection of increasingly resistant strains.

Sequence variation of necdin gene in Bovidae.

BACKGROUND: Necdin (NDN), a member of the melanoma antigen family showing imprinted pattern of expression, has been implicated as causing Prader-Willi symptoms, and known to participate in cellular growth, cellular migration and differentiation. The region where NDN is located has been associated to QTLs affecting reproduction and early growth in cattle, but location and functional analysis of the molecular mechanisms have not been established. METHODS: Here we report the sequence variation of the entire coding sequence from 72 samples of cattle, yak, buffalo, goat and sheep, and discuss its variation in Bovidae. Median-joining network analysis was used to analyze the variation found in the species. Synonymous and non-synonymous substitution rates were determined for the analysis of all the polymorphic sites. Phylogenetic analysis were carried out among the species of Bovidae to reconstruct their relationships. RESULTS: From the phylogenetic analysis with the consensus sequences of the studied Bovidae species, we found that only 11 of the 26 nucleotide changes that differentiate them produced amino acid changes. All the SNPs found in the cattle breeds were novel and showed similar percentages of nucleotides with non-synonymous substitutions at the N-terminal, MHD and C-terminal (12.3, 12.8 and 12.5%, respectively), and were much higher than the percentage of synonymous substitutions (2.5, 2.6 and 4.9%, respectively). Three mutations in cattle and one in sheep, detected in heterozygous individuals were predicted to be deleterious. Additionally, the analysis of the biochemical characteristics in the most common form of the proteins in each species show very little difference in molecular weight, pI, net charge, instability index, aliphatic index and GRAVY (Table 4) in the Bovidae species, except for sheep, which had a higher molecular weight, instability index and GRAVY. CONCLUSIONS: There is sufficient variation in this gene within and among the studied species, and because NDN carry key functions in the organism, it can have effects in economically important traits in the production of these species. NDN sequence is phylogenetically informative in this group, thus we propose this gene as a phylogenetic marker to study the evolution and conservation in Bovidae.

Conservation of Repeats at the Mammalian KCNQ1OT1-CDKN1C Region Suggests a Role in Genomic Imprinting.

KCNQ1OT1 is located in the region with the highest number of genes showing genomic imprinting, but the mechanisms controlling the genes under its influence have not been fully elucidated. Therefore, we conducted a comparative analysis of the KCNQ1/KCNQ1OT1-CDKN1C region to study its conservation across the best assembled eutherian mammalian genomes sequenced to date and analyzed potential elements that may be implicated in the control of genomic imprinting in this region. The genomic features in these regions from human, mouse, cattle, and dog show a higher number of genes and CpG islands (detected using cpgplot from EMBOSS), but lower number of repetitive elements (including short interspersed nuclear elements and long interspersed nuclear elements), compared with their whole chromosomes (detected by RepeatMasker). The KCNQ1OT1-CDKN1C region contains the highest number of conserved noncoding sequences (CNS) among mammals, where we found 16 regions containing about 38 different highly conserved repetitive elements (using mVista), such as LINE1 elements: L1M4, L1MB7, HAL1, L1M4a, L1Med, and an LTR element: MLT1H. From these elements, we found 74 CNS showing high sequence identity (>70%) between human, cattle, and mouse, from which we identified 13 motifs (using Multiple Em for Motif Elicitation/Motif Alignment and Search Tool) with a significant probability of occurrence, 3 of which were the most frequent and were used to find transcription factor-binding sites. We detected several transcription factors (using JASPAR suite) from the families SOX, FOX, and GATA. A phylogenetic analysis of these CNS from human, marmoset, mouse, rat, cattle, dog, horse, and elephant shows branches with high levels of support and very similar phylogenetic relationships among these groups, confirming previous reports. Our results suggest that functional DNA elements identified by comparative genomics in a region densely populated with imprinted mammalian genes may be related to the regulation of imprinted gene expression.

Molecular evolution of type II MAGE genes from ancestral MAGED2 gene and their phylogenetic resolution of basal mammalian clades.

Type II melanoma-associated antigens (MAGE) are a subgroup of about a dozen proteins found in various locations in the genome and expressed in normal tissues, thus are not related to cancer as the type I MAGE genes. This gene family exists as a single copy in non-mammals and monotremata, but found as two copies in metatherians and occur as a diverse group in all eutherians. Our studies suggest MAGED2 as the ancestor of this subfamily and the most likely evolutionary history of eutherian type II MAGE genes is hereby proposed based on synteny conservation, phylogenetic relations, genome location, homology conservation, and the protein and gene structures. Type II genes can be divided into two: those with 13 exons (MAGED1, MAGED2, TRO, and MAGED4) and those with only one exon (MAGEE1, MAGEE2, MAGEF1, NSMCE3, MAGEH1, MAGEL2, and NDN) with different evolutionary patterns. Our results suggest a need to change the gene nomenclature to MAGE1 (the ancestral gene), currently designated as LOC103095671 and LOC100935086, in opossum and Tasmanian devil, respectively, and MAGE2 (the duplicated one), currently designated as LOC100617402 and NDNL2, respectively, to avoid confusion. We reconstructed the phylogenetic relationships among 23 mammalian species using the combined sequences of MAGED1, MAGED2, MAGEL2, and NDN, because of their high divergence, and found high levels of support, being able to resolve the phylogenetic relationships among Euarchontoglires, Laurasiatheria, Afrotheria, and Xenarthra, as an example that small, but phylogenetically informative sequences, can be very useful for resolving basal mammalian clades.

Identificación de Escherichia coli enteropatógena en niños con síndrome diarreico agudo del Estado Sucre, Venezuela / Identification of enteropathogenic Escherichia coli in children with acute diarrheic syndrome from Sucre State, Venezuela

Introducción. Escherichia coli es uno de los principales agentes causales del síndrome diarreico agudo. Objetivo. Identificar grupos clonales de E. coli enteropatógena en 485 casos de diarrea aguda en niños entre 0 y 10 años de edad atendidos en centros de salud de los municipios de Arismendi, Benítez y Sucre del estado Sucre, Venezuela, entre marzo y diciembre de 2011. Materiales y métodos. Previo consentimiento informado, se recolectaron muestras fecales y se identificó E. coli mediante coprocultivo estándar y serología con antisueros polivalentes y monovalentes. Se aisló el ADN y se amplificaron los genes eae (intimina) y bfpA (bundlina) mediante dos pruebas de reacción en cadena de la polimerasa (PCR) múltiples. Resultados. En 39,6 % de los coprocultivos se determinó la presencia de infección bacteriana. La prevalencia de E. coli fue de 54,7 %; 82,9 % de estas cepas fue positivo por serología para los serogrupos y el serotipo evaluados, principalmente en niños entre los 0 y los 2 años (37,9 %). El 48,6 % de las cepas de E. coli amplificaron para el gen eae y, de estas, 58,8 % se clasificó como cepas de E. coli enteropatógena típica (eae+ y bfp+). El ECEP II fue el serogrupo más frecuente (38,7 %), con predominio de bacterias E. coli enteropatógenas típicas (60 %). El alelo ß de la intimina fue el más identificado (74,5 %) en las cepas positivas para el gen eae. Solo se identificaron cuatro cepas con el serotipo O157:H7 utilizando antisueros, las cuales no amplificaron mediante PCR para los genes eae y bfpA. Conclusiones. Este estudio demostró la importancia de aplicar pruebas moleculares en la identificación de las cepas de E. coli causantes de diarrea de diversa gravedad.

Introduction: Diarrheagenic Escherichia coli is an important causative agent of acute diarrheic syndrome. Objective: To identify clonal groups of enteropathogenic E. coli (EPEC), in 485 children with acute diarrhea aged 0 to 10 years attending health care centers in Arismendi, Benítez and Sucre municipalities, Sucre state, Venezuela, from March to December, 2011. Materials and methods: After obtaining the informed consent, stool samples were collected. Escherichia coli was identified using standard coproculture methods and serology with polyvalent and monovalent antisera. DNA was isolated, and eae (intimin) and bfpA (bundlin) genes were amplified through two multiplex polymerase chain reactions (PCR). Results: The presence of bacterial infection was determined in 39.6% of coprocultures. The prevalence of E. coli was 54.7%; 82.9% of these isolates were positive by serology for the evaluated serogroups and serotypes, which were mostly identified in children between 0 and 2 years (37.9%); 48.6% of E. coli strains amplified the eae gene; of these, 58.8% were classified as typical EPEC (eae+ y bfp+). EPEC II was the most common serogroup (38.7%), with predominance of typical EPEC (60%). In positive strains for eae gene, the ß intimin allele was the most frequently identified (74.5%). Only four strains with O157:H7 serotype were identified, which showed no PCR amplification of the eae and bfpA genes. Conclusion: This study showed the importance of molecular tests to identify diarrheagenic E. coli strains causing clinical conditions of varying severity.

Molecular identification of multidrug resistant Enterobacter hormaechei in Venezuela / Identificación molecular de Enterobacter hormaechei multidrogoresistente en Venezuela

Besides the importance of Enterobacter cloacae species complex as a nosocomial pathogen, little is known about the frequency of each species/genotype. Here, we describe a strain of E. hormaechei subsp. hormaechei isolated from a bronchial secretion of a patient, in the Intensive Care Unit at the General Hospital of Cumaná, Venezuela, who died due to complications of his infection. The molecular identification was done by sequencing the 16S rRNA gene and comparing it to sequences from the GenBank. This strain showed resistance to multiple families of antibiotics (MDR), and the genes blaKPC and blaVIM were detected by PCR. This is the first time E. hormaechei has been identified in Venezuela.

A pesar de la importancia de las especies del complejo Enterobacter cloacae como patógeno nosocomial, poco se conoce sobre la frecuencia de cada especie/genotipo. Aquí se describe una cepa de E. hormaechei subsp. hormaechei aislada de una secreción bronquial de un paciente internado en la Unidad de Cuidados Intensivos del Hospital General de Cumaná, Venezuela, quien murió producto de complicaciones de su infección. La identificación molecular fue hecha por secuenciación del gen ARNr 16S y porcomparación con las secuencias del GenBank. Esta cepa mostró resistencia a múltiples familias de antibióticos (MDR) y se detectaron los genes blaKPCyblaVIMpor PCR. Este es el primer reporte de E. hormaechei en Venezuela.

Identification of enteropathogenic Escherichia coli in children with acute diarrheic syndrome from Sucre State, Venezuela.

INTRODUCTION: Diarrheagenic Escherichia coli is an important causative agent of acute diarrheic syndrome.  OBJECTIVE: To identify clonal groups of enteropathogenic E. coli (EPEC), in 485 children with acute diarrhea aged 0 to 10 years attending health care centers in Arismendi, Benítez and Sucre municipalities, Sucre state, Venezuela, from March to December, 2011.  MATERIALS AND METHODS: After obtaining the informed consent, stool samples were collected. Escherichia coli was identified using standard coproculture methods and serology with polyvalent and monovalent antisera. DNA was isolated, and eae (intimin) and bfpA (bundlin) genes were amplified through two multiplex polymerase chain reactions (PCR).  RESULTS: The presence of bacterial infection was determined in 39.6% of coprocultures. The prevalence of E. coli was 54.7%; 82.9% of these isolates were positive by serology for the evaluated serogroups and serotypes, which were mostly identified in children between 0 and 2 years (37.9%); 48.6% of E. coli strains amplified the eae gene; of these, 58.8% were classified as typical EPEC (eae+ y bfp+). EPEC II was the most common serogroup (38.7%), with predominance of typical EPEC (60%). In positive strains for eae gene, the ß intimin allele was the most frequently identified (74.5%). Only four strains with O157:H7 serotype were identified, which showed no PCR amplification of the eae and bfpA genes.  CONCLUSION: This study showed the importance of molecular tests to identify diarrheagenic E. coli strains causing clinical conditions of varying severity.

Identificación de genes que codifican enzimas modificadoras de aminoglucósidos en cepas intrahospitalarias de Klebsiella pneumoniae / Identification of genes encoding aminoglycoside-modifying enzymes of Klebsiella pneumoniae nosocomial isolates

Las infecciones por Klebsiella pneumoniae, constituyen un problema creciente en los centros hospitalarios. El objetivo de la presente investigación fue evaluar la resistencia a los aminoglucósidos, así como la presencia de genes que codifican enzimas modificadoras de aminoglucósidos (EMA) en aislados intrahospitalarios de Klebsiella pneumoniae. Se analizaron 56 cepas provenientes de pacientes con diagnóstico de infección intrahospitalaria del Hospital Universitario “Antonio Patricio de Alcalá”, durante el periodo enero-septiembre de 2008. Se determinó la susceptibilidad antimicrobiana mediante los métodos de difusión y dilución en agar, siguiendo los lineamientos del Instituto de Estándares Clínicos y de Laboratorio. Se empleó la técnica de la reacción en cadena de la polimerasa (PCR) para detectar los genes que codifican EMA. Se encontró resistencia a gentamicina y tobramicina en el 33,9% y 35,7%, respectivamente. Los fenotipos de resistencia a aminoglucósidos más frecuentes fueron I (ANGMKTob) y II (GMKTob). Se identificaron los genes aadA (21,4%), aac(3)-IIa (16,1%), aadB (14,3%), aac (6`)-Ib (3,6%) y aph (3`)-Ia (1,8%). En 10 cepas se observó la presencia de más de un gen y en 13 cepas se correlacionó el fenotipo con los genes encontrados. La resistencia a los aminoglucósidos en los aislados evaluados se debe, principalmente, a enzimas de tipo acetiltransferasas.

Klebsiella pneumoniae infection is a growing problem in hospitals. The objective of this study was to evaluate resistance to aminoglycosides and detection of genes encoding for aminoglycoside modifying enzymes (AME) in hospital isolates of K. pneumoniae. Fifty-six isolates from patients with diagnosis of nosocomial infection at the University Hospital Antonio Patricio de Alcala, during the period January to September 2008 were included for study. Antimicrobial susceptibility was determined by the methods of diffusion and agar dilution, following the Institute for Clinical and Laboratory Standards Guidelines. Genes encoding AME were determined by the polymerase chain reaction procedure. Resistance results for Gentamycin were 33.9% and for Tobramycin 35.7%. Aminoglycoside resistance phenotypes most frequently identified were I (ANGMKTob) and II (GMKTob). The genes involved were aadA (21.4%), aac(3)-IIa (16.1%), aadB (14.3%), aac (6`)-Ib (3.6%) y aph (3`)-Ia (1.8%) For 10 of the isolates studied more than one gene was identified. In 13 isolates the phenotype corresponded to the genes found. Aminoglycoside resistance in the isolates studied is mainly due to the presence of acetyltransferase enzymes.

AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA.

The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America.

Population Structure Shapes Copy Number Variation in Malaria Parasites.

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

Molecular identification of multidrug resistant Enterobacter hormaechei in Venezuela.

Besides the importance of Enterobacter cloacae species complex as a nosocomial pathogen, little is known about the frequency of each species/genotype. Here, we describe a strain of E. hormaechei subsp. hormaechei isolated from a bronchial secretion of a patient, in the Intensive Care Unit at the General Hospital of Cumaná, Venezuela, who died due to complications of his infection. The molecular identification was done by sequencing the 16S rRNA gene and comparing it to sequences from the GenBank. This strain showed resistance to multiple families of antibiotics (MDR), and the genes blaKPC and blaVIM were detected by PCR. This is the first time E. hormaechei has been identified in Venezuela.