Phylo-geographic analysis of whitefly on the basis of mitochondrial cytochrome oxidase 1 gene / Análise filogeográfica da mosca-branca com base no gene citocromo oxidase 1 mitocondrial

Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).

Bemisia tabaci é um complexo de espécies que causa danos a uma ampla gama de hospedeiros vegetais por meio de alimentação séria. Ele transmite vírus de plantas de diferentes grupos para importantes safras agrícolas. Algumas safras comerciais importantes do Paquistão são cana-de-açúcar, arroz, tabaco e óleo de semente. Apresenta alta variabilidade genética e é diferenciado em raças ou biótipos. Os biótipos são: biótipo Q, biótipo B, biótipo B2, biótipo M, biótipo L, biótipo A, biótipo H, biótipo C, biótipo K, biótipo N, biótipo R, biótipo E, biótipo P, biótipo J, biótipo S, biótipo AN . Embora o relatório atual baseado no estudo bayesiano das sequências de DNA do gene 1 da oxidase do citocromo mitocondrial (CO1) tenha classificado as diferentes populações de moscas-brancas em doze grupos genéticos, que são Mediterrâneo, África Subsaariana com folha de prata, Oceano Índico, Ásia II, Ásia I, Austrália, Novo Mundo, Itália, China, África Subsaariana sem folha prateada, Batata-doce Mediterrâneo / Ásia Menor / África e Uganda. Os begomovírus são o maior grupo de vírus transmitidos por B. tabaci e causam as principais doenças de culturas, como a doença do cacho do tomate e da pimenta-malagueta, doença do mosaico da mandioca, doença do mosaico amarelo de leguminosas e doença do enrolamento da folha do algodão. O principal objetivo do presente estudo é inculpar conhecimento sobre a diversidade genética da mosca-branca em campos de algodão em todo o Paquistão por meio da análise da sequência parcial de DNA do gene mitocondrial Citocromo Oxidase I (mtCO1).

Phylo-geographic analysis of whitefly on the basis of mitochondrial cytochrome oxidase 1 gene

Abstract Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).

Resumo Bemisia tabaci é um complexo de espécies que causa danos a uma ampla gama de hospedeiros vegetais por meio de alimentação séria. Ele transmite vírus de plantas de diferentes grupos para importantes safras agrícolas. Algumas safras comerciais importantes do Paquistão são cana-de-açúcar, arroz, tabaco e óleo de semente. Apresenta alta variabilidade genética e é diferenciado em raças ou biótipos. Os biótipos são: biótipo Q, biótipo B, biótipo B2, biótipo M, biótipo L, biótipo A, biótipo H, biótipo C, biótipo K, biótipo N, biótipo R, biótipo E, biótipo P, biótipo J, biótipo S, biótipo AN . Embora o relatório atual baseado no estudo bayesiano das sequências de DNA do gene 1 da oxidase do citocromo mitocondrial (CO1) tenha classificado as diferentes populações de moscas-brancas em doze grupos genéticos, que são Mediterrâneo, África Subsaariana com folha de prata, Oceano Índico, Ásia II, Ásia I, Austrália, Novo Mundo, Itália, China, África Subsaariana sem folha prateada, Batata-doce Mediterrâneo / Ásia Menor / África e Uganda. Os begomovírus são o maior grupo de vírus transmitidos por B. tabaci e causam as principais doenças de culturas, como a doença do cacho do tomate e da pimenta-malagueta, doença do mosaico da mandioca, doença do mosaico amarelo de leguminosas e doença do enrolamento da folha do algodão. O principal objetivo do presente estudo é inculpar conhecimento sobre a diversidade genética da mosca-branca em campos de algodão em todo o Paquistão por meio da análise da sequência parcial de DNA do gene mitocondrial Citocromo Oxidase I (mtCO1).

Discovery, structure and function of plasmid mediated shufflon / 生物工程学报

Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of Salmonella typhi. The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene rci, and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene mcr-1, which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of mcr-1. Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.

A soil bacterial catabolic pathway on the move: Transfer of nicotine catabolic genes between Arthrobacter genus megaplasmids and invasion by mobile elements

The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolicpathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotineto nicotine blue, a-ketoglutarate and succinate. Various modules of these genes have been shown to be presentin gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bpplasmid pZXY21 of Arthrobacter sp. ZXY2 (96% to 100% at the nucleotide level) permitted the identificationof the limits of this DNA fragment. At the 50 end of the nic-genes are located the ORFs of two predictedintegrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of asmall transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C ofStaphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolictransposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encodingtransposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide thenic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes canbe mobilized and spread by horizontal gene transfer to other soil bacteria.

High plasmid variability, and the presence of IncFIB, IncQ, IncA/C, IncHI1B, and IncL/M in clinical isolates of Klebsiella pneumoniae with bla KPC and bla NDM from patients at a public hospital in Brazil

Abstract INTRODUCTION Antibiotic resistance in carbapenemase-producing Klebsiella pneumoniae is acquired and disseminated mainly by plasmids. Therefore, we aimed to investigate the occurrence of carbapenemase genes, analyze the genetic diversity by ERIC-PCR, and examine the most common plasmid incompatibility groups (Incs) in clinical isolates of K. pneumoniae from colonization and infection in patients from a hospital in Brazil. METHODS Twenty-seven isolates of carbapenem-resistant K. pneumoniae were selected and screened for the presence of carbapenemase genes and Incs by PCR, followed by amplicon sequencing. RESULTS The bla KPC and bla NDM genes were detected in 24 (88.8 %) and 16 (59.2 %) of the isolates, respectively. Thirteen isolates (48.1 %) were positive for both genes. The IncFIB (92.6 %) and IncQ (88.8 %) were the most frequent plasmids, followed by IncA/C, IncHI1B, and IncL/M, indicating that plasmid variability existed in these isolates. To our knowledge, this is the first report of IncHI1B in Brazil. We found eight isolates with clonal relationship distributed in different sectors of the hospital. CONCLUSIONS The accumulation of resistance determinants, the variability of plasmid Incs, and the clonal dissemination detected in K. pneumoniae isolates demonstrate their potential for infection, colonization, and the dissemination of different resistance genes and plasmids.

Study on the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer / 中国基层医药

Objective@#To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.@*Methods@#SGC-7901 cells were selected from gastric cancer cell lines, and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT-PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments, the expression of llinc00052 and the effect on Wnt/β-catenin expression were investigated to explore whether llinc00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt/β-catenin signaling pathway.@*Results@#Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10±4.29), and the number of transmembrane cells in the overexpressed llinc000522 plasmid group was (169.24±6.99)(t=8.956, P=0.001). The scratch test showed that the migration distance in the llinc000522 overexpression transfection plasmid group was significantly higher than that in the no-load plasmid transfection group(r=0.907, P<0.01). The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75±6.32)% vs.(73.34±9.14)%](t=5.998, P<0.05). Compared with the transfection of blank plasmid, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up-regulated(P<0.05), while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1, Wnt3a, Wnt2, and β-catenin mRNA were significantly increased [(1.82±0.11), (1.52±0.15), (1.42±0.21), (1.71±0.19)](P<0.05), but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P<0.05), while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1, Wnt3a, Wnt2 and β-catenin were also significantly increased[(1.53±0.09), (1.4±0.21), (1.33±0.07), (1.47±0.19)](P<0.05), but their expressions were still lower than those of the gene transfected with llinc000522 alone.@*Conclusion@#In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt/β-catenin pathway.

Study on the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer / 中国基层医药

Objective To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.Methods SGC-7901 cells were selected from gastric cancer cell lines ,and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT -PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments ,the expression of llinc00052 and the effect on Wnt／β-catenin expression were investigated to explore whether llinc 00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt／β-catenin signaling pathway.Results Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10 ±4.29),and the number of transmembrane cells in the overexpressed llinc 000522 plasmid group was (169.24 ±6.99)(t＝8.956,P＝0.001). The scratch test showed that the migration distance in the llinc 000522 overexpression transfection plasmid group was significantly higher than that in the no -load plasmid transfection group (r＝0.907,P＜0.01).The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75 ±6.32)% vs.(73.34 ±9.14)%] (t＝5.998,P＜0.05).Compared with the transfection of blank plasmid,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up -regulated(P＜0.05),while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1,Wnt3a,Wnt2,and β-catenin mRNA were significantly increased [(1.82 ± 0.11),(1.52 ±0.15),(1.42 ±0.21),(1.71 ±0.19)] ( P＜0.05),but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection ,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P＜0.05),while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1,Wnt3a,Wnt2 and β-catenin were also significantly increased [(1.53 ±0.09),(1.4 ±0.21), (1.33 ±0.07),(1.47 ±0.19)](P＜0.05),but their expressions were still lower than those of the gene transfected with llinc000522 alone.Conclusion In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt ／β-catenin pathway.

Staphylococcus aureus procedentes de quesos costeños de Valledupar; susceptibilidad a antibióticos y perfil plasmídico / Staphylococcus aureus from Valledupar cheeses; susceptibility to antibiotics and plasmid profile

Resumen En este estudio se investigó la susceptibilidad a antibióticos y el perfil plasmídico de S aureus aislado de queso costeño, blando, semiduro y duro, expendidas en diferentes puntos de venta de la ciudad de Valledupar. Por el método de Difusión del disco en agar, se determinó la resistencia a antibióticos y con la técnica de lisis alcalina y electroforesis en gel de agarosa, el perfil plasmídico. Como resultado, se obtuvo una carga microbiana por encima de 103 UFC/g, que está sobre el valor promedio máximo permitido, según la norma covenin 1538-92, el cual indica que se puede desencadenar brotes por intoxicación con estafilocócos. Se demostró la presencia de S. aureus en los quesos costeños blandos (75%), seguidos por los quesos semiduros (25%) ambos de origen (artesanal), los cuales son de alto consumo en Valledupar. Se establecieron 4 patrones diferentes de resistencia en las cepas de S. aureus aisladas de los quesos, siendo el patrón TER común para dos cepas, el resto de los patrones fueron únicos (PR, CR y EI). Una cepa fue resistente a P, productora de β-lactamasas, su CMI más alta fue 32µg/ml; todas las cepas mostraron sensibilidad a oxacilina, gentamicina, ciprofloxacina, cefoxitin, clindamicina, trimetoprim sulfa, vancomicina, rifampin, e imipenen. Hubo bandas plasmídicas con tamaños de 23kb encontrándose cepas con 1 plásmidos.

Abstract In this study, the antibiotic susceptibility was investigated and the plasmid profile of S aureus isolated from coastal cheese, soft, semi-hard and hard, expended in different outlets of the city of Valledupar. For the method of diffusion of the agar disk, the antibiotic resistance was determined and with the technique of alkaline lysis and electrophoresis in the agarose gel, the plasmid profile. As a result, was obtained one microbial load, above of 103 UFC/g, which is on average the maximum allowed value, according to the standard covenin 1538-92, which indicates that it can trigger outbreaks by Staphylococcal poisoning. The presence of S.aureus in coastal soft cheese was shown (75%), followed by semi-hard cheeses (25%) both home (handmade), which are of high consume in Valledupar. four different resistance patterns were established in the strains of the S.aureus isolated from the cheeses, being TER the common pattern for five strains, the rest of the patterns were unique (PR, CR and EI). One strain was resistant to P, producer of the β-lactamases, the CMI more tall was 32µg/ml; All the strains show sensibility to oxacillin, gentamycin, ciprofloxacin, cephoxitin, clindamycin, trimetoprim sulfa, vancomycin, rifampin, and imipenem. There plasmid bands with sizes between 23kb, being 1 strains with plasmids.

Detection of multidrug-resistant Enterobacteriaceae isolated from river waters flowing to the Guanabara Bay and from clinical samples of hospitals in Rio de Janeiro, Brazil / Detección de enterobacterias multirresistentes aisladas en aguas de los ríos que desembocan en la bahía de Guanabara y en muestras de hospitales de Río de Janeiro, Brasil

Abstract Introduction: The use of antibiotics in humans, animal husbandry and veterinary activities induces selective pressure leading to the colonization and infection by resistant strains. Objective: We evaluated water samples collected from rivers of the Guanabara Bay, which have suffered minor and major environmental degradation, and clinical samples of hospital origin to detect evidence of the presence of resistance genes to aminoglycosides, beta-lactam antibiotics and fluoroquinolones in strains of Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and Escherichia coli. Materials and methods: For isolation of the water strains we employed culture media containing 32 μg/ml cephalotin and 8 μg/ml gentamicin. The strains from clinical materials were selected using culture media containing 8 μg/ml gentamicin. The strains were identified and subjected to antimicrobial susceptibility testing (AST), plasmid DNA extraction and polymerase chain reaction (PCR) to detect genes encoding enzymes modifying aminoglycosides (EMA), extended-spectrum beta-lactamases (ESBL) and plasmid mechanisms of quinolone resistance (PMQR). Results: The AST of the isolates recovered from water samples showed multidrug-resistance profiles similar to those found in isolates recovered from clinical materials. All isolates from water samples and 90% of the isolates from clinical samples showed at least one plasmid band. In the PCR assays, 7.4% of the isolates recovered from water samples and 20% of those from clinical materials showed amplification products for the three antimicrobial classes. Conclusion: We believe that the detection of microorganisms presenting genetic elements in environments such as water is necessary for the prevention and control of their dissemination with potential to infect humans and other animals in eventual contact with these environments.

Resumen Introducción. El uso de antibióticos en seres humanos, en la industria pecuaria y en las actividades veterinarias induce una presión selectiva que resulta en la colonización e infección con cepas resistentes. Objetivo. Determinar la presencia de genes de resistencia a aminoglucósidos, betalactámicos y fluoroquinolonas en cepas de Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae y Escherichia coli, obtenidas de muestras de agua de los ríos que desembocan en la bahía de Guanabara y de muestras clínicas de hospitales de Río de Janeiro. Materiales y métodos. En la selección de las cepas resistentes obtenidas de las muestras de agua de los ríos, se emplearon medios de cultivo que contenían 32 μg/ml de cefalotina y 8 μg/ ml de gentamicina. En el caso de las muestras de especímenes clínicos, se usaron medios de cultivo que contenían 8 μg/ml de gentamicina. Las cepas se identificaron y se sometieron a pruebas de sensibilidad antimicrobiana, extracción de ADN plasmídico y pruebas de reacción en cadena de la polimerasa (PCR) para detectar los genes que codifican aquellas enzimas que modifican los aminoglucósidos, las betalactamasas de espectro extendido (BLEE) y los mecanismos de resistencia a las quinolonas mediados por plásmidos. Resultados. Se encontraron perfiles de resistencia a los antimicrobianos similares en los dos grupos. En todas las bacterias obtenidas de las muestras de agua y en 90 % de las muestras clínicas, se evidenciaron bandas de plásmidos asociados con la transferencia de genes de resistencia. En las pruebas de PCR, se obtuvieron productos de amplificación de los genes de resistencia para las tres clases de antimicrobianos analizados, en el 7,4 % de las bacterias recuperadas de las muestras de agua y en el 20 % de aquellas recuperadas de las muestras clínicas. Conclusión. La detección de microorganismos con elementos genéticos que confieren resistencia a los antibióticos en ambientes como el agua, es una estrategia necesaria para prevenir y controlar la diseminación de estos agentes patógenos con potencial para infectar a humanos y a otros animales en dichos ambientes.

Plasmid profiles among some ESKAPE pathogens in a tertiary care centre in south India

Background & objectives: Plasmid has led to increase in resistant bacterial pathogens through the exchange of antimicrobial resistance (AMR) genetic determinants through horizontal gene transfer. Baseline data on the occurrence of plasmids carrying AMR genes are lacking in India. This study was aimed to identify the plasmids associated with AMR genetic determinants in ESKAPE pathogens. Methods: A total of 112 ESKAPE isolates including Escherichia coli (n=37), Klebsiella pneumoniae (n=48, including 7 pan-drug susceptible isolates), Acinetobacter baumannii (n=8), Pseudomonas aeruginosa (n=1) and Staphylococcus aureus (n=18) were analyzed in the study. Isolates were screened for antimicrobial susceptibility and whole genome sequencing of isolates was performed using Ion Torrent (PGM) sequencer. Downstream data analysis was done using PATRIC, ResFinder, PlasmidFinder and MLSTFinder databases. All 88 whole genome sequences (WGS) were deposited at GenBank. Results: Most of the study isolates showed resistant phenotypes. As analyzed from WGS, the isolates included both known and unknown sequence types. The plasmid analysis revealed the presence of single or multiple plasmids in the isolates. Plasmid types such as IncHI1B(pNDM-MAR), IncFII(pRSB107), IncFIB(Mar), IncFIB(pQil), IncFIA, IncFII(K), IncR, ColKP3 and ColpVC were present in K. pneumoniae. In E. coli, IncFIA, IncFII, IncFIB, Col(BS512), IncL1, IncX3 and IncH were present along with other types. S. aureus harboured seven different plasmid groups pMW2 (rep 5), pSAS1 (rep 7), pDLK1 (rep 10), pUB110 (rep US12), Saa6159 (rep 16), pKH12 (rep 21) and pSA1308 (rep 21). The overall incidence of IncF type plasmids was 56.5 per cent followed by Col type plasmids 18.3 per cent and IncX 5.3 per cent. Other plasmid types identified were <5 per cent. Interpretation & conclusions: Results from the study may serve as a baseline data for the occurrence of AMR genes and plasmids in India. Information on the association between phenotypic and genotypic expression of AMR was deciphered from the data. Further studies on the mechanism of antibiotic resistance dissemination are essential for enhancing clinical lifetime of antibiotics.

Caracterización de cepas clínicas y ambientales de Salmonella enterica subsp. enterica serovar Heidelberg aisladas en Chile / Characterization of Salmonella Heidelberg strains isolated in Chile

Background: Salmonella Heidelberg (S. Heidelberg) causes gastroenteritis and sometimes bacteremia and endocarditis. In other countries, this serovar has multidrug resistance including extended-spectrum β-lactamases (ESBLs) and AmpC (β-lactamases (AmpC), associated with the blaCMY-2 gene. In Chile, an outbreak by S. Heidelberg occurred in 2011, the phenotypic and genetic characteristics of Chilean strains are unknown. Aim: To determine the antimicrobial susceptibility, presence of plasmids and virulence factor genes in S. Heidelberg strains isolated in Chile over the period 2006-2011. Material and Methods: In sixty-one S. Heidelberg clinical and environmental strains collected by the Public Health Institute in Chile during 2006-2011, antimicrobial susceptibility, plasmids and virulence factor genes (invA, sifA, pefA, agfA, lpfA and, stkD) were studied. Results: S. Heidelberg had a high susceptibility to sulfamethoxazole-trimethoprim, gentamicin, ceftriaxone, ceftiofur, chloramphenicol, amoxicillin-clavulanic acid and ampicillin. However, 52% had decreased susceptibility to ciprofloxacin and 33% resistance to tetracycline. ESBLs were detected in three strains isolated from blood cultures, environment and human feces. The latter strain was positive for AmpC and blaCMY-2 gene. Fifty three of 61 strains showed one to seven plasmids of 0.8 to approximately 30 kb. Most plasmids were small with sizes between 0.8 and 2 kb. All isolates were positive for all genes except pefA. Conclusions: S. Heidelberg isolated from Chilean samples was susceptible to first-line antimicrobials, except tetracycline and ciprofloxacin. The emergence of strains with ESBLs and AmpC should be a warning. The strains were homogeneous for virulence genes, but heterogeneous in their plasmids.

Cell Death Mechanisms in Esophageal Squamous Cell Carcinoma Induced by Vesicular Stomatitis Virus Matrix Protein

OBJECTIVES: Vesicular stomatitis virus (VSV) is under development as an oncolytic virus due to its preferential replication in cancer cells and oncolytic activity, however the viral components responsible have not yet been determined. In this study the effects of VSV wild-type (wt) and M51R-mutant matrix proteins (M51R-mMP) on apoptosis, pyroptosis, necroptosis, and autophagy pathways, in an esophagus cancer cell line (KYSE-30) were investigated. METHODS: The KYSE-30 cells were transfected with pcDNA3.1 plasmids encoding wt or M51R-mMP, and apoptosis, pyroptosis, necroptosis, and autophagy were evaluated 48 and 72 hours after transfection. RESULTS: KYSE-30 cells transfected with VSV wt and M51R-mMPs significantly reduced cell viability to < 50% at 72 hours post-transfection. M51R-MP significantly increased the concentration of caspase-8 and caspase-9 at 48 and 72 hours post-transfection, respectively ( p < 0.05). In contrast, no significant changes were detected following transfection with the VSV wt plasmid. Moreover, VSV wt and M51R-mMP transfected cells did not change the expression of caspase-3. VSV wt and M51R-mMPs did not mMP change caspase-1 expression (a marker of pyroptosis) at 48 and 72 hours post-transfection. However, M51R-mMP and VSV wt transfected cells significantly increased RIP-1 (a marker of necroptosis) expression at 72 hours post-infection ( p < 0.05). Beclin-1, a biomarker of autophagy, was also induced by transfection with VSV wt or M51R-mMPs at 48 hours post-transfection. CONCLUSION: The results in this study indicated that VSV exerts oncolytic activity in KYSE-30 tumor cells through different cell death pathways, suggesting that M51R-mMP may potentially be used to enhance oncolysis.

Petroleum contamination and bioaugmentation in bacterial rhizosphere communities from Avicennia schaueriana

ABSTRACT Anthropogenic activity, such as accidental oil spills, are typical sources of urban mangrove pollution that may affect mangrove bacterial communities as well as their mobile genetic elements. To evaluate remediation strategies, we followed over the time the effects of a petroleum hydrocarbon degrading consortium inoculated on mangrove tree Avicennia schaueriana against artificial petroleum contamination in a phytoremediation greenhouse experiment. Interestingly, despite plant protection due to the inoculation, denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene fragments amplified from the total community DNA indicated that the different treatments did not significantly affect the bacterial community composition. However, while the bacterial community was rather stable, pronounced shifts were observed in the abundance of bacteria carrying plasmids. A PCR-Southern blot hybridization analysis indicated an increase in the abundance of IncP-9 catabolic plasmids. Denaturing gradient gel electrophoresis of naphthalene dioxygenase (ndo) genes amplified from cDNA (RNA) indicated the dominance of a specific ndo gene in the inoculated petroleum amendment treatment. The petroleum hydrocarbon degrading consortium characterization indicated the prevalence of bacteria assigned to Pseudomonas spp., Comamonas spp. and Ochrobactrum spp. IncP-9 plasmids were detected for the first time in Comamonas sp. and Ochrobactrum spp., which is a novelty of this study.

Asociación de la resistencia al mercurio con la resistencia a antibióticos en Escherichia coli aislados del litoral de Lima, Perú / Association of mercury resistance with resistance to antibiotics in Escherichia coli isolated from the coast of Lima, Peru

El objetivo de este estudio fue investigar la resistencia al mercurio y los antibióticos, y la transferencia de resistencia al mercurio por plásmidos conjugativos en 55 cepas de Escherichia coli aisladas de aguas superficiales del litoral de Lima, Perú. Se determinó la Concentración Mínima Inhibitoria (CMI) a diversos antibióticos y al mercurio en las cepas aisladas. Para confirmar la resistencia plasmídica al mercurio, se realizó la curación de estos con dodecil sulfato de sodio (SDS) 10%. El ensayo de transferencia de plásmidos por conjugación se realizó usando la cepa receptora E. coli DH5α sólo con las cepas que mostraron sensibilidad al mercurio después de la curación. La extracción de los plásmidos de resistencia fue realizada sólo en las cepas transconjugantes resistentes al mercurio. 41 (74.5%) cepas fueron resistentes al mercurio (HgR), presentando CMIs entre 30 μM (8.25 ppm) y 300 μM (82.5 ppm), de estás, 33 fueron HgR mediante plásmidos y de este último grupo, 14 fueron también resistentes a antibióticos. Sólo 6 cepas poseían plásmidos conjugativos con resistencia al mercurio, mostrando una frecuencia de transconjugación entre 9.41x10-4 y 4.76x10-2%. La alta prevalencia de cepas de E. coli HgR aisladas de la costa limeña podría ser un problema de salud pública y ambiental. En este sentido, los plásmidos congugativos pueden contribuir con la diseminación de mercurio y/o resistencia a antibióticos entre comunidades bacterianas en ambientes marinos.

The Lima coast is highly affected by anthropogenic effluents from wastewater from contaminated urban rivers that flow into the coast. The objective of this study was to investigate the resistance to mercury and antibiotics, and the transfer of resistance to mercury by conjugative plasmids in 55 strains of Escherichia coli isolated of surface seawater from coastal Lima, Peru. The Minimum Inhibitory Concentration (MIC) was determined for various antibiotics and for mercury in the isolated strains. To confirm the plasmid resistance to mercury, the curing was carried out with 10% sodium dodecyl sulfate (SDS). The plasmid transfer assay by conjugation was performed using the E. coli DH5α as recipient strain only with the strains that showed sensitivity to mercury after curing. The extraction of the resistance plasmids was carried out only in the transconjugant strains resistant to mercury. 41 (74.5%) strains were resistant to mercury (HgR), with MICs between 30 μM (8.25 ppm) and 300 μM (82.5 ppm), of these, 33 were HgR by plasmids and of this last group, 14 were also resistant to antibiotics. Only 6 strains had conjugative plasmids with mercury resistance, showing a transconjugation frequency between 9.41x10-4 and 4.76x10-2%. The high prevalence of HgR in E. coli strains isolated from the coast of Lima could be a public and environmental health problem. In this sense, congugative plasmids can contribute to the spread of mercury and/or resistance to antibiotics among bacterial communities in marine environments.

Determinación de perfiles plasmídicos de Escherichia coli y Klebsiella pneumoniae productoras de ß-lactamasas de espectro extendido aisladas en urocultivos / Plasmid profile determination of Escherichia coli and Klebsiella pneumoniae producers of extended spectrum ß-lactamase isolated in urocultives

Introducción. El aumento de las tasas de resistencia en bacterias uropatógenas y su propagación se han convertido en un problema de salud pública a nivel mundial, cobrando mayor importancia la diseminación de aislamientos resistentes productores de ß-lactamasas de espectro extendido, resultando útil la caracterización molecular de plásmidos y otros elementos genéticos de las bacterias para establecer la relación genética que existe entre ellas y tener datos relevantes que puedan sugerir la dirección de propagación de este tipo de resistencia. El objetivo de nuestra investigación fue determinar los perfiles plasmídicos en aislamientos de Escherichia coli y Klebsiella pneumoniae productoras de ß-lactamasas de espectro extendido de urocultivos del Instituto Nacional de Salud del Niño, Lima, Perú. Métodos. Estudio descriptivo, observacional de corte transversal. Resultados. Se determinaron diferentes patrones electroforéticos, obteniéndose de 1 hasta 7 bandas por muestra y en total 15 bandas de distinto tamaño, variando desde 1.5 kb hasta más de20 kb. Teniendo en cuenta los patrones de huella obtenidos, los aislamientos estudiados se distribuyeron en diferentes grupos o ramas, usando el programa NTSYSpc 2.1 y el algoritmo de agrupamiento UPGMA. Conclusión. No se encontró similitud genética marcada entre los aislamientos de origen comunitario y de origen asociado a atención sanitaria, por lo que no se puede establecer una relación significativa y determinar un origen común entre los mismos.

Introduction. The increase in resistance rates in uropathogenic bacteria and their spread has become in a significant worldwide public health, becoming more important the spread of resistant isolates producing of extended spectrum ß-lactamases, which are useful the molecular characterization of plasmids and other genetic elements of the bacteria to establish the genetic relationship between them, to have relevant data that we can suggest the direction of propagation of this type of resistance. The object of research was to determine the plasmid profiles in isolates of Escherichia coli and Klebsiella pneumoniae producing of extended spectrum ß-lactamases from urine cultures of the National Institute of Child Health, Lima, Peru. Methods. Descriptive, observational cross-sectional study. Results. Were detected different electrophoretic patternsin the samples, obtained from 1-7 bands per sample and a total of 15 bands of different sizes, which varied from 1.5 kb to more than 20 kb. Considering the fingerprint patterns, the isolates analyzed were distributed into different clusters or branches, using the NTSYSpc 2.1 program and the UPGMA clustering algorithm. Conclusions. No genetic similarity was found between isolates of community origin and of origin associated with health care, so it is not possible to establish a meaningful relationship and to determine a common origin among them.

Estudo piloto sobre imunomodulação na leishmaniose visceral murina: bloqueio da sinalização de IL-10 e uso de IL-1 / Pilot study on immunomodulation in murine visceral leishmaniasis: blockade of IL-10 signaling and use of IL-12

INTRODUÇÃO: A leishmaniose visceral (LV) é uma doença infecto-parasitária e é considerada negligenciada, segundo a organização mundial de saúde. O cão e o homem fazem parte da manutenção do ciclo da infecção. O crescente aumento de animais infectados acarreta em um problema de saúde pública, pois representa eminente risco de infeção para os humanos. Na LV, a resposta imune celular é suprimida favorecendo a replicação do parasito nos macrófagos e a exacerbação da doença. Estudos apontam que esta supressão pode estar relacionada à secreção da interleucina-10 (IL-10), pois esta tem o papel de regular o excesso de resposta inflamatória, inibindo a produção de IL-12. OBJETIVO: Nesta pesquisa, propõese produzir e purificar plasmídeos codificando receptor de IL-10 e IL-12 murinos, bem como produzir e purificar o receptor de IL-10 murino na forma solúvel no sistema baculovíruscélulas de inseto, visando avaliar os efeitos de substâncias imunomoduladoras em camundongos infectados com Leishmania infantum/chagasi, com o intuito de desenvolver um método imunoterápico para cães com LV. METODOLOGIA: Na primeira parte deste trabalho, camundongos BALB/c foram infectados com L. infantum/chagasi, por via venosa e em seguida foram injetados quatro vezes, com intervalo de 7 dias entre as doses, com salina (G1), pcDNA3.1 (plasmídeo controle) (G2), pcDNA3.1mIL-12 (G3), pcDNA3.1mIL-10R1 (G4) e pcDNA3.1mIL-12+ pcDNA3.1mIL-10R1 (G5), por via intramuscular, seguida de eletroporação. Os efeitos da administração dos plasmídeos foram avaliados através da análise histológica do fígado e baço e pela determinação da carga parasitária do baço por PCR em tempo real. Na segunda parte deste trabalho foi produzida e purificada através do sistema baculovírus-célula de inseto, a proteína rmusIL-10R1. Além disso, foi realizado o ensaio de atividade biológica em células MC/9 para avaliar se a proteína produzida e purificada possui atividade funcional. RESULTADOS: A administração de plasmídeos codificadores do receptor de IL-10R1 em associação com IL-12 seguida de eletroporação apresentou efeitos sistêmicos nos grupos G3 e G5, como presença de granulomas maduros no parênquima hepático e nos espaços portais, intenso infiltrado inflamatório no parênquima hepático, aumento do centro germinativo no tecido esplênico e redução da carga parasitária. O rendimento obtido da proteína produzida e purificada foi de 678 µg por litro de cultivo. CONCLUSÃO: Os efeitos observados na primeira parte do trabalho podem ser atribuídos somente à ação da IL-12 e não do receptor de IL-10. Na segunda etapa do trabalho evidenciou-se que a proteína rmusIL-10R1 não foi capaz de bloquear a sinalização mediada por IL-10 murina

INTRODUCTION: Visceral leishmaniasis (VL) is an infectious-parasitic disease and is considered neglected, according to the World Health Organization. The dog and the man are part of maintaining the cycle of infection. The increasing numbers of infected animals have a public health problem, as it represents an imminent risk of infection for humans. In LV, the cellular immune response is suppressed favoring the replication of the parasite in the macrophages and the exacerbation of the disease. Studies indicate that this suppression may be related to the secretion of interleukin-10 (IL-10), since it has the role of regulating the excess inflammatory response, inhibiting the production of IL12. PURPOSE: In this research, it is proposed to produce and purify murine IL-10 and IL-12 receptor encoding plasmids, as well as to produce and purify the murine IL-10 receptor in the soluble form of the baculovirus-insect cells system, in order to evaluate the effects of immunomodulatory substances in mice infected with Leishmania infantum / chagasi, with the aim of developing an immunotherapeutic method for dogs with VL. METHODOLOGY: In the first part of this work, BALB / c mice were infected with venom intravenous L. infantum and then injected four times, with 7 days between doses, with saline (G1), pcDNA3.1 (G2), pcDNA3.1mIL-12 (G3), pcDNA3.1mIL-10R1 (G4) and pcDNA3.1mIL-12 + pcDNA3.1mIL-10R1 (G5), followed by electroporation. The effects of plasmid administration were assessed by histological analysis of the liver and spleen and determination of spleen parasite load by real-time PCR. In the second part of this work the rmusIL-10R1 protein was produced and purified through the baculovirus-insect cell system. In addition, the biological activity assay was performed on MC / 9 cells to assess whether the produced and purified protein has functional activity. RESULTS: Administration of IL-10R1 receptor-encoding plasmids in association with IL-12 followed by electroporation showed systemic effects in the G3 and G5 groups, such as presence of mature granulomas in the hepatic parenchyma and portal spaces, intense inflammatory infiltrate in the hepatic parenchyma , increase of the germinal center in the splenic tissue and reduction of the parasitic load. The yield of the produced and purified protein was 678 µg per liter of culture. CONCLUSION: The effects observed in the first part of the work can be attributed only to the action of IL-12 and not the IL-10 receptor. In the second stage of the work it was shown that the rmusIL-10R1 protein was not able to block murine IL-10 mediated signaling.

Caracterización de los plásmidos de cepas de escherichia coli aislados de coprocultivos de niños sanos / Characterization of plasmids of escherichia coli strains isolated from stool culture of healthy children

La microbiota intestinal representa una reserva potencial de organismos resistentes a los antimicrobianos, y el sitio donde los genes de resistencia pueden ser transferidos desde la microbiota comensal a los microorganismos virulentos. En este trabajo se caracterizaron los perfiles fenotípicos de resistencia a diversos agentes antimicrobianos, en aislados de Escherichia coli, obtenidos de niños sanos, menores de 5 años de edad, y la capacidad de transmisibilidad de esos determinantes de resistencia, mediante ensayos de conjugación. Los aislados de E. coli se obtuvieron partir de coprocultivos de niños sanos mediante el uso de placas de Mc Conkey suplementadas con ampicilina y se les determinó el perfil de resistencia a diversos antibióticos, para luego realizar ensayos de conjugación. A partir de 90 coprocultivos, fueron aisladas 33 cepas de E. coli resistentes a algún antibiótico, presentándose un 66,6% del total de las cepas resistentes en al menos dos antibióticos. Luego de los ensayos de conjugación, se encontró que un 47,4% de las cepas presenta plásmidos conjugativos, transfiriendo marcadores de resistencia. Los patrones generados por enzimas de restricción fueron distintos entre ellos. Estos resultados nos permiten sugerir que estos elementos extracromosomales sean los responsables de la rápida diseminación de la resistencia a los antimicrobianos en la población bacteriana de niños sanos.

Gastrointestinal microbiota represents the potential reserve of antimicrobial-resistant organisms, and the site where resistance genes can be transferred from the commensally microbiota to virulent microorganisms. In this work we characterized the phenotypic resistance profiles to various antimicrobial agents in strains of Escherichia coli isolated from healthy children, less than 5 years of age, and the ability of these determinants of resistance to be mobilized by conjugation. The isolation of E. coli strains from stool culture from healthy children was made through the use of Mc Conkey media supplemented with ampicillin. The profile of resistance to various antibiotics was determined and then conjugation was carried out. From 90-stool culture 33 strains of E. coli resistant to some antibiotic were isolated, 63.6% of bacteria were resistant to -at least- two antibiotic. It have be demonstrated that 47.4% of the isolates harbored conjugative plasmids, which can mobilize markers of resistance. Restriction profiles analysis showed that all patterns were different. These results allow us to suggest that these extracromosomals elements are responsible for the rapid spread of resistance to antimicrobials in the bacterial population of healthy children.

Influence of antibiotic concentration on the integration frequency of resistance cassette in the integron / 中华检验医学杂志

Objective@#To observe the integration frequency of aadA2 resistance cassette at attI site of the integron under different concentration of streptomycin. @*Methods@#Class 1 integron with known gene sequence was cloned into plasmid pACYC184 to produce recombinant plasmid pACIDA, meanwhile the integrase gene was cloned into plasmid pET28a to construct recombinant plasmid pETINT. These two recombinant plasmids were consecutively transformed into E. coli BL(DE3). These transformed bacteria was cultured in the LB medium at 37 ℃ overnight with addition of different concentration of streptomycin. The copy number of total integrons and the copy number of integrated aadA2 at attI site of integrons were determined by using real-time PCR. and the integration frequency is the result of the former divided by the latter. @*Results@#The resulting frequencies were (1.97±0.24)×10-3, (3.23±1.77)×10-3, (3.27±0.67)×10-3, 0.45±0.13 and 1.32±0.11, with respective streptomycin concentrations of 0, 20, 30, 40 and 50 μg/ml. The background frequency of integration without integrase overexpression was less than (1.75±0.33)×10-7. @*Conclusion@#These findings indicate that antibiotic concentration significantly increase recombination frequency of aadA2 resistance cassette at attI site of the integron, catalyzed by integron integrase.(Chin J Lab Med, 2018, 41: 531-535)

Development and applications of external quality control materials for MTHFR 677C/T genotypes / 中华检验医学杂志

Objective To evaluate the performance of MTHFR 677 genotyping external quality assessment ( EQA) program using plasmid DNA constructed in vitro as quality control samples and discuss the problems in clinical laboratories enrolled in the program .Methods Recombinant plasmid carrying MTHFR 677C locus sequence was constructed as wild type sample and plasmid with MTHFR 677T mutation was generated with site-directed mutagenesis as mutant type sample .Heterozygous mutant samples were obtained after equal proportion of the two plasmids .EQA scheme were held twice a year in 2016 and 2017, and sample panels contained 5 different samples using recombinant plasmid DNA containing all types of MTHFR 677 locus genotypes.Participating laboratories were asked to test samples using their routine methods and report the results before deadlines .26, 28, 52 and 56 effective reports were received respectively in the four EQA schemes .The scores of each lab were calculated based on their results and the overall compliance of different samples as well as the sensitivity and specificity of different methods were calculated using Microsoft Excel .Results MTHFR 677 locus genotypes of the constructed plasmid were verified by Sanger sequencing and there was no failure of sample detection in the four EQA schemes , which suggest that the plasmid has good clinical applicability .About 96.15％( 25/26 ) ,100％( 28/28 ) ,96.15％ (50/52)and 98.21％(55/56) of the laboratories submitted correct results for all samples in the four EQA schemes.The overall compliance rate were 99.23％ ( 129/130 ) , 100％( 140/140 ) ,96.92％ ( 252/260 ) and 98.93％(277/280) respectively.All laboratories using digital FISH and microarrays got full marks in four EQA schemes.The compliance rates for fluorescent PCR were 97.5％ ( 39/40 ) , 100％ ( 45/45 ) , 94.29％(66/70) and 100％ (95/95) respectively, while the rates were 100％ (20/20), 100％ (15/15), 90％(36/40) and 92.5％(37/40) for Sanger sequencing.Conclusions The recombinant plasmid DNA constructed in this study can effectively detect the performance of reagents with good clinical applicability.The results of EQA programs suggested that the overall accuracy rate of laboratories enrolled was high enough , while some laboratories′performance still needs to be improved .Quality controls in clinical laboratories were essential to assure the accuracy of results .

Cloning,expression and biological activity of m6A binding protein YTHDF2 / 军事医学

Objective To construct the recombinant plasmid of YTH domain family 2(YTHDF2)and express it in E.coli in order to obtain YTHDF2 fusion protein that was capable of binding m 6A-modified RNA.Methods The coding region of YTHDF2 gene was amplified by RT-PCR.The recombinant plasmid pET-28a-YTHDF2 was constructed and expressed in E.coli.The fusion protein was purified by Ni2+-NTA resin affinity chromatography, while the fusion protein activity was analyzed by Ni2+-NTA magnetic spheres.Results and Conclusion The recombinant YTHDF2 protein was expressed in E.coli BL21(DE3)and purified.YTHDF2 fusion protein was capable of binding RNA with m 6A-modification. The preparation of YTHDF2 fusion protein provides an essential tool to study the biological function of RNA with m6A-modification.