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Description of the subject. In aquatic environments, prolonged exposure to heavy metals and/or antimicrobials selects for resistant bacteria, increasing the health risk for the population.Objectives. To evaluate antimicrobial and arsenic (As) resistance, presence of addiction systems and biofilm production in Gram-negative bacilli isolated from the Zimapan dam.Method. Surface water was sampled from 10 sites in the western area of the dam. CHROMagar™ ESBL and mSuperCARBA™ plates were used to isolate fermenting (FGB) and non-fermenting (NFGB) Gram-negative bacilli resistant to beta-lactams and carbapenems, respectively. Resistance was verified by the agar-diffusion method and double-disk synergy test. Genes for extended spectrum beta-lactamases (ESBL), carbapenemases and addiction systems were detected by PCR. Biofilm production and resistance to As and/or cefotaxime (CTX) were measured by spectrophotometry. The tolerance As was determined by serial dilution in LB agar adding variable concentrations of Na3AsO2.Results. Of 47 beta-lactams resistant strains, 77.5% produced ESBL, with blaCTX-M (38% and 62%) and blaTEM-2 (41% and 51%) genes being detected in pisciculture and recreational areas, respectively. Significant resistance to imipenem and meropenem was detected, although no carbapenemase genes were detected; relE, pnd, ccdA, and vagC addiction system genes were the most frequent. Nineteen strains showed resistance up to 183 ppm of Na3AsO2, 11 strains at 400 ppm and 3 strains up to 2,000 ppm. Biofilm production was detected in 83% of the strains. Conclusions. Contamination of the Zimapan dam with multidrug-resistant, biofilm-forming, As- and CTX-tolerant bacteria with different plasmid addiction systems requires urgent prevention and environmental control strategies. Barrage de Zimapan au Mexique, réservoir de bactéries productrices de bêta-lactamases à spectre étendu et de résistance à l'arsenicDescription du sujet. En milieu aquatique, une exposition prolongée aux métaux lourds et/ou aux antimicrobiens sélectionne des bactéries résistantes, augmentant ainsi le risque pour la santé de la population.Objectifs. Évaluer la résistance aux antimicrobiens et à l'arsenic (As), la présence de systèmes de dépendance et la production de biofilm dans les bacilles à Gram-négatif isolés du barrage de Zimapan.Méthode. Des échantillons d'eau de surface ont été prélevés sur 10 sites dans la zone ouest du barrage. Des plaques CHROMagar™ ESBL et mSuperCARBA™ ont été utilisées pour isoler des bacilles à Gram-négatif fermentant (FGB) et non fermentant (NFGB) résistants aux bêta-lactamines et aux carbapénèmes, respectivement. La résistance a été vérifiée par la méthode de diffusion sur gélose et le test de synergie double disque. Les gènes des bêta-lactamases à spectre étendu (BLSE), des carbapénémases et des systèmes de dépendance ont été détectés par la réaction en chaîne par polymérase. La production de biofilm et la résistance à l'As et/ou au céfotaxime (CTX) ont été mesurées par spectrophotométrie. La tolérance à l’As a été déterminée par des dilutions en série sur de la gélose LB en ajoutant diverses concentrations de Na3AsO2.Résultats. Sur 47 souches résistantes aux bêta-lactamines, 77,5 % produisaient des BLSE, les gènes blaCTX-M (38 % et 62 %) et blaTEM-2 (41 % et 51 %) étant détectés respectivement dans les zones de pisciculture et de loisirs. Une résistance significative à l’imipénème et au méropénème a été détectée, bien qu’aucun gène de carbapénémase n’ait été détecté. Les gènes du système addictif relE, pnd, ccdA et vagC étaient les plus fréquents. Dix-neuf souches ont montré une résistance jusqu'à 183 ppm de Na3AsO2, 11 souches jusqu'à 400 ppm et 3 souches jusqu'à 2 000 ppm. La production de biofilm a été détectée dans 83 % des souches.Conclusions. La contamination du barrage de Zimapan par des bactéries multirésistantes, formant un biofilm, tolérantes à l'As et au CTX avec différents systèmes de dépendance aux plasmides nécessite des stratégies urgentes de prévention et de contrôle environnemental.

Background Ctis globally the most common cause of sexually transmitted infections. A new variant of ct with a deletion in the cryptic plasmid has been found in Sweden, following an unexpected 25% increase in genital infections in 2006. This variant escapes routine diagnostic PCR-tests. Thus a new nuclear acid amplification test (NAAT), which uses the cryptic plasmid as well as the MOMP-gene as target area was developed. The MOMP-gene encodes a protein (OMP-1) which represents 60% of the proteins embedded in the peptidoglycans of the bacterial cell wall. The aim of this study was to define the number of cryptic plasmid-/MOMP+ patients. Methods Between 2009 to 2012 we analysed probes of 11250 individuals (patients and controls) processing the ProbeTecET® test (BD, USA). Of these 407 showed a positive result and were treated according to current guidelines. 33 patients tested negative, however, reported a persistence of discomfort such as burning sensations in the urethra, urethral discharge and occasionally conjunctivitis. These patients were additionally tested with the GenoQuick® CT (HAIN Lifescience, Germany), which specifically and simultaneously detects both, the MOMP-gene and the cryptic plasmide. Material was taken from urethral, cervical, rectal, pharyngeal, conjunctival smears and from the Douglas-space. Results All 33 patients tested positive when processing the GenoQuick® CT. Thus 7.5% of infected patients were only identified processing an additional detection set. Conclusion In our centre 7.5% of ct infected patients were tested “false negative” when only the cryptic plasmide was analysed. These 33 patients were identified processing a more sensitive test system and subsequently were treated.

The yeast non-Mendelian factor [psi+] has been suggested to be a self-modified protein analogous to mammalian prions. Here it is reported that an intermediate amount of the chaperone protein Hsp104 was required for the propagation of the [psi+] factor. Overproduction or inactivation of Hsp104 caused the loss of [psi+]. These results suggest that chaperone proteins play a role in prion-like phenomena, and that a certain level of chaperone expression can cure cells of prions without affecting viability. This may lead to antiprion treatments that involve the alteration of chaperone amounts or activity

Abstract Staphylococcus aureus enterotoxin D is one of the serotypes most commonly associated with food poisoning. Further characterization of the enterotoxin D-encoding plasmid revealed the presence of an open reading frame which encodes a previously unidentified enterotoxin, designated staphylococcal enterotoxin J (SEJ). SEJ is a protein of 269 amino acid residues which has substantial sequence similarity to the staphylococcal A, E, D family of enterotoxins. The enterotoxin D and J open reading frames are transcribed in opposite directions and are separated by an 895 nucleotide intergenic region which contains a perfect inverted repeat, with each arm of the repeat having a length of 21 nucleotides. Chloramphenicol acetyl transferase (cat) transcriptional fusions were used to quantify expression from the enterotoxin gene promoters. Both enterotoxin genes are expressed in S. aureus. However, only sed is regulated by the agr virulence gene signal transduction pathway. Western blot analyses utilizing anti-enterotoxin antisera have confirmed the results obtained with the cat reporter system. PCR amplification studies suggest that the sej determinant may be present on all sed-encoding plasmids.

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.

To initiate defense responses against invasion of pathogenic organisms, animals and plants must recognize microbe-associated molecular patterns (MAMPs). In this study, the elicitor activity of bacterial DNA on the model plant Arabidopsis thaliana was examined. EcoRI-digested plasmid DNA induced defense responses such as generation of reactive oxygen species and deposition of callose, whereas SmaI- and HapII-digested plasmid DNA and EcoRI-digested herring DNA did not remarkably induce these responses. Further, methylation of the CpG sequence of plasmid DNA and Escherichia coli DNA reduced the level of the defense responses. The endocytosis inhibitors wortmannin and amantadine significantly inhibited DNA-induced defense responses. These results suggest that nonmethylated CpG DNA, as a MAMP, induced defense responses in Arabidopsis and that non-methylated DNA seems to be translocated into the cytoplasm by endocytosis.

Sphingobium chungbukense DJ77 is capable of metabolizing priority chemicals of human health concern such as polycyclic aromatic hydrocarbons (PAHs), extracellular polysaccharide (EPS), and antibiotics. Here, we report the complete DNA and genetic organization of the plasmid pSY2 from strain DJ77. A DNA sequence analysis revealed that pSY2 comprises 18,779 bp encoding 22 open reading frames (ORFs) with 59.5% G+C content. The ORFs on pSY2 were classified into DNA replication, conjugative function, transposition, plasmid stability/partition, and other functional groups (transport, fatty acid biosynthesis, stress, and growth rate regulation). Three ORFs on pSY2 were hypothetical proteins.

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5/K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5/K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5/K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4

Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods, and they are regarded as potential food-grade hosts for protein expression. The aim of this study was to develop a broad-host-range shuttle vector for the genetic study and biotechnological evaluation of this genus by using a Leuconostoc-derived plasmid. A cryptic plasmid, pCB18, was obtained from Leuconostoc citreum CBNU75; its nucleotide sequence was 1,821 bp long and had only 39.2% G+C content. A Leuconostoc-Escherichia coli shuttle vector, pLeuCM, was constructed by combining pCB18 and pEK104, and it was successfully replicated in both E. coli and L. citreum. The shuttle vector was replicated by following the rolling circle replication mechanism, and it showed over 80% segregational stability after 100 generations of cell division. The β-galactosidase gene of Lactobacillus plantarum was subcloned into pLeuCM, and this construct was successfully expressed in L. citreum. The pLeuCM plasmid was replicated in L. citreum, L. mesenteroides, Lb. plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. These results demonstrate that pLeuCM can be used as a potential genedelivery tool for many lactic acid bacteria.

The flanking genetic structure of qnrA1 in Korean Enterobacter cloacae was identical to that of the Chinese Escherichia coli strain, the first qnrA1-carrying strain reported in Asia. Analysis of restriction enzyme sites and Southern blot hybridization results showed that qnrA1 was transferred between E. cloacae and E. coli via Inc HI2 type plasmid.