Plasmid profiles and antibiotic resistance of Pseudomonas aeruginosa isolated from different clinical samples in Jordan

Aims@#The aim of this study was to investigate plasmid profiles and antibiotic resistance patterns of multidrug resistant Pseudomonas aeruginosa strains isolated from different clinical specimens in Jordan.@*Methodology and results@#Pseudomonas aeruginosa strains were isolated from different clinical specimens from different hospitals and primary health care centers. The antimicrobial susceptibility of P. aeruginosa isolates was determined using the disc diffusion method against 16 commonly used antimicrobial drugs. Plasmid DNAs were extracted from lysed P. aeruginosa cells using the plasmid alkaline lysis method and visualized using agarose gel electrophoresis followed by ethidium bromide staining. The isolated strains of P. aeruginosa were resistant to cefepime (90%), meropenem (70%), ceftazidime (60%), piperacillin (55%), aztreonam (50%), ciprofloxacin and tobramycin (35%), gentamicin (29%), imipenem and amikacin (20%). All the isolates were sensitive to colistin. Plasmid analysis of the clinical isolates showed the presence of 0 to 3 plasmids with a size range of 1 to 25 kb compared to the standard strain of Escherichia coli (ATCC 25922).@*Conclusion, significance and impact of study: @#The results obtained in this study showed some correlation between the patterns of antibiotic resistance and plasmid profiles.

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).

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).

Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile

Background The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. Results Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM−1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st-5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. Conclusions We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

Mecanismos de resistencia a la colistina, la nitrofurantoína y la fosfomicina en Enterobacterales / Mechanisms of resistance to colistin, nitrofurantoin and fosfomycin in Enterobacteriaceae

Resumen Las enterobacterias son un grupo amplio y heterogéneo de bacilos Gram negativos que se aíslan de forma rutinaria en el laboratorio clínico y se asocian a una gran cantidad de cuadros clínicos. Aquellas resistentes a antibióticos de última línea, como a los carbapenémicos, representan un gran reto en los centros de salud. Ante la dificultad para tratar infecciones causadas por este tipo de bacterias, se ha retomado el uso de antimicrobianos clásicos como la colistina, la nitrofurantoína y la fosfomicina. El objetivo de este trabajo es detallar los principales mecanismos de resistencia para estos tres fármacos descritos en enterobacterias. Para ello, se efectuó una revisión bibliográfica de artículos científicos publicados entre los años 1999 y 2022, utilizando las bases de datos PubMed (NCBI), PLOS, Redalyc, Google Scholar y Science Direct. En este proceso, se usaron las palabras clave "Carbapenem-Resistant Enterobacteriaceae", "colistin", nitrofurantoin", "fosfomycin", "resistance" y "plasmids". Se encontró que los mecanismos de resistencia son variados y abarcan fenómenos como modificación del sitio blanco, inactivación enzimática, impermeabilidad y eflujo. Además, los determinantes genéticos de resistencia se encuentran en cromosomas o en plásmidos. Conocer este tipo de información permite mejorar la vigilancia basada en el laboratorio, combatir el problema de resistencia a los antimicrobianos y optimizar el uso de estos antibióticos que forman parte del escaso arsenal para el tratamiento de ciertas infecciones causadas por microorganismos multidrogorresistentes.

Abstract Enterobacteriaceae is a large and heterogeneous group of Gram-negative bacilli that are routinely isolated in the clinical laboratory and are associated with a large number of clinical conditions. Those resistant to last-line antibiotics, such as carbapenems, represent a great challenge in health-care centers. Given the difficulty in treating this type of infections, the use of old drugs such as colistin, nitrofurantoin and fosfomycin has been studied. The objective of this work is to detail the main resistance mechanisms described in Enterobacteriaceae for these three antibiotics. To do this, a survey of scientific articles from the years 1999 to 2022 was carried out using databases such as PubMed (NCBI), Google Scholar, PLOS, Redalyc and Science Direct. In this process, keywords "Carbapenem- Resistant Enterobacteriaceae", "colistin", nitrofurantoin", "fosfomycin", "resistance" and "plasmids" were used. Resistance mechanisms were found to be varied and involve phenomena such as target site modification, enzyme inactivation, impermeability, and efflux. In addition, the genetic determinants of resistance are found at the chromosomal level or in plasmids. Knowing this type of information makes it possible to improve laboratory-based surveillance, fight the problem of resistance to antibiotics and take care of these antibiotics, which are part of the scarce arsenal for the treatment of certain infections caused by multidrug-resistant microorganisms.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.