Search

Integrons are prokaryotic genetic elements known to carry and exchange antibiotic resistance gene cassettes through a site-specific recombinase called integrase. In this work, 107 Aeromonas isolates from environmental origin, including fish, water, and sediments, were investigated for the presence of integrons. Using specific primers for Class 1, 2 and 3 integrases, only Class 1 and Class 2 integrons were detected. Detection of Class 2 integrases and their associated variable regions required two rounds of polymerase chain reaction (PCR). Sequencing of the intI2 amplicons confirmed them as integrase-derived products. Class 1 integrons were detected in 26 out of 107 isolates. PCR amplification of the variable regions associated to these integrons revealed an outstanding homogeneity, 25 of them having variable regions with an identical dfrA12-orfF-aadA2 cassette array and one integron carrying only the dfrA16 cassette. To assess clone diversity, chromosomal DNA from isolates was subjected to enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), which discarded clonality in all instances. Class 2 integrons were surprisingly more prevalent than Class1 integrons, being detected in 60 out of 107 isolates. Forty-six of them showed a unique ERIC profile, while the remaining 14 strains displayed profiles that could be grouped in five different patterns. Cassette arrangements of all Class 2 variable regions were those described as the most prevalent (dfrA1-sat2-aadA1). A rather startling result of this work is the sensitivity to trimethoprim, streptomycin, and streptothricin of most strains, despite the presence of the cognate resistance genes. To know the integron distribution in environmental Aeromonas species, a phylogenetic reconstruction was done using rpoD/gyrB or rpoD/gyrA gene sequences. Isolates bearing these elements corresponded to Aeromonas hydrophila, Aeromonas veronii, Aeromonas salmonicida, Aeromonas dhakensis, Aeromonas sanarellii, Aeromonas taiwanensis, Aeromonas media, Aeromonas caviae, Aeromonas jandaei, and Aeromonas sp. This work revealed an unusual high incidence of Class 2 integrons and a low variability of cassette arrangements in environmental Aeromonas species.

The increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. There are currently some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9) demonstrated 71% inhibitory activity of QS at 100 μM (IC50 = 90.9 μM) in Chromobacterium violaceum, a Gram-negative bacterium. The aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13–15) and three 2-oxazolines (16–18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. The binding mode of the test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-pentyloxyphenyl-2-imidazoline (13) reduced the production of violacein (IC50 = 56.38 μM) without affecting bacterial growth, suggesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C6-AHL) on the CviR protein. Ten amino acid residues in the active binding site of C6-AHL in the Cvi receptor interacted with 13, and 7 of these are the same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and 9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C6-AHL for the orthosteric site.

This article describes, for the first time, the role of the nasal mucosa (NM) as the initial site for the Histoplasma capsulatum mycelial-to-yeast transition. The results highlight that yeasts may arrive to the cervical lymph nodes (CLN) via phagocytes. Bats and mice were intranasally infected with H. capsulatum mycelial propagules and they were killed 10, 20, and 40 minutes and 1, 2, and 3 hours after infection. The NM and the CLN were monitored for fungal presence. Yeasts compatible with H. capsulatum were detected within the NM and the CLN dendritic cells (DCs) 2-3 hours postinfection, using immunohistochemistry. Histoplasma capsulatum was re-isolated by culturing at 28°C from the CLN of both mammalian hosts 2-3 hours postinfection. Reverse transcription-polymerase chain reaction assays were designed to identify fungal dimorphism, using mycelial-specific (MS8) and yeast-specific (YPS3) gene expression. This strategy supported fast fungal dimorphism in vivo, which began in the NM 1 hour postinfection (a time point when MS8 and YPS3 genes were expressed) and it was completed at 3 hours (a time point when only the YPS3 transcripts were detected) in both bats and mice. The presence of intracellular yeasts in the nasal-associated lymphoid tissue (NALT), in the NM nonassociated with the NALT, and within the interdigitating DCs of the CLN suggests early fungal dissemination via the lymph vessels.

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.

Quorum sensing is a communication system among bacteria to sense the proper time to express their virulence factors. Quorum sensing inhibition is a therapeutic strategy to block bacterial mechanisms of virulence. The aim of this study was to synthesize and evaluate new bioisosteres of N-acyl homoserine lactones as Quorum sensing inhibitors in Chromobacterium violaceum CV026 by quantifying the specific production of violacein. Five series of compounds with different heterocyclic scaffolds were synthesized in good yields: thiazoles, 16a&ndash, c, thiazolines 17a&ndash, c, benzimidazoles 18a&ndash, c, pyridines 19a&ndash, c and imidazolines 32a&ndash, c. All 15 compounds showed activity as Quorum sensing inhibitors except 16a. Compounds 16b, 17a&ndash, c, 18a, 18c, 19c and 32b exhibited activity at concentrations of 10 &micro, M and 100 &micro, M, highlighting the activity of benzimidazole 18a (IC50 = 36.67 &micro, M) and 32b (IC50 = 85.03 &micro, M). Pyridine 19c displayed the best quorum sensing inhibition activity (IC50 = 9.66 &micro, M). Molecular docking simulations were conducted for all test compounds on the Chromobacterium violaceum CviR protein to gain insight into the process of quorum sensing inhibition. The in-silico data reveal that all 15 the compounds have higher affinity for the protein than the native AHL ligand (1). A strong correlation was found between the theoretical and experimental results.

In 1995, Pseudomonas sp. ADP, capable of metabolizing atrazine, was isolated from contaminated soil. Genes responsible for atrazine mineralization were found scattered in the 108.8 kb pADP-1 plasmid carried by this strain, some of them flanked by insertion sequences rendering them unstable. The goal of this work was to construct a transcriptional unit containing the atz operon in an easy to transfer manner, to be introduced and inherited stably by Gram-negative bacteria. atz genes were PCR amplified, joined into an operon and inserted onto the mobilizable plasmid pBAMD1–2. Primers were designed to add efficient transcription and translation signals. Plasmid bearing the atz operon was transferred to different Gram-negative strains by conjugation, which resulted in Tn5 transposase-mediated chromosomal insertion of the atz operon. To test the operon activity, atrazine degradation by transposants was assessed both colorimetrically and by high-performance liquid chromatography (HPLC). Transposants mineralized atrazine more efficiently than wild-type Pseudomonas sp. ADP and did not accumulate cyanuric acid. Atrazine degradation was not repressed by simple nitrogen sources. Genes conferring atrazine-mineralizing capacities were stable and had little or null effect on the fitness of different transposants. Introduction of catabolic operons in a stable fashion could be used to develop bacteria with better degrading capabilities useful in bioremediation.

Bark beetles from Dendroctonus genus promote ecological succession and nutrient cycling in coniferous forests. However, they can trigger outbreaks leading to important economic losses in the forest industry. Conifers have evolved resistance mechanisms that can be toxic to insects but at the same time, bark beetles are capable of overcoming tree barriers and colonize these habitats. In this sense, symbiont yeasts present in the gut of bark beetles have been suggested to play a role in the detoxification process of tree defensive chemicals. In the present study, genes related to this process were identified and their response to a terpene highly toxic to bark beetles and their symbionts was analyzed in the Cyberlindnera americana yeast. The genome and transcriptome of C. americana (ChDrAdgY46) isolated from the gut of Dendroctonus rhizophagus were presented. Genome analysis identified 5752 protein-coding genes and diverse gene families associated with the detoxification process. The most abundant belonged to the Aldo-Keto Reductase Superfamily, ATP-binding cassette Superfamily, and the Major Facilitator Superfamily transporters. The transcriptome analysis of non-α-pinene stimulated and α-pinene stimulated yeasts showed a significant expression of genes belonging to these families. The activities demonstrated by the genes identified as Aryl-alcohol dehydrogenase and ABC transporter under (+)-α-pinene suggest that they are responsible, that C. americana is a dominant symbiont that resists high amounts of monoterpenes inside the gut of bark beetles.

The fungicide carbendazim is an ecotoxic agent affecting aquatic biota. Due to its suspected hormone-disrupting effects, it is considered a "priority hazard substance" by the Water Framework Directive of the European Commission, and its degradation is of major concern. In this work, a horizontal tubular biofilm reactor (HTBR) operating in plug-flow regime was used to study the kinetics of carbendazim removal by an acclimated microbial consortium. The reactor was operated in steady state continuous culture at eight different carbendazim loading rates. The concentrations of the fungicide were determined at several distances of the HTBR. At the loading rates tested, the highest instantaneous removal rates were observed in the first section of the tubular biofilm reactor. No evidence of inhibition of the catabolic activity of the microbial community was found. Strains of the genera Flectobacillus, Klebsiella, Stenotrophomonas, and Flavobacterium were identified in the biofilm; the last three degrade carbendazim in axenic culture.

With the increasing antibiotic resistance of bacterial strains, alternative methods for infection control are in high demand. Quorum sensing (QS) is the bacterial communication system based on small molecules. QS is enables bacterial biofilm formation and pathogenic development. The interruption of QS has become a target for drug discovery, but remains in the early experimental phase. In this study, we synthesized a set of six compounds based on a scaffold (alkyl-quinoxalin-2(1H)-one), new in the anti-QS of Gram-negative bacteria Aeromonas caviae Sch3. By quantifying biofilm formation, we were able to monitor the effect of these compounds from concentrations of 1 to 100 µM. Significant reduction in biofilm formation was achieved by 3-hexylylquinoxalin-2(1H)-one (11), 3-hexylylquinoxalin-2(1H)-one-6-carboxylic acid (12), and 3-heptylylquinoxalin-2(1H)-one-6-carboxylic acid (14), ranging from 11% to 59% inhibition of the biofilm. This pilot study contributes to the development of anti-QS compounds to overcome the clinical challenge of resistant bacteria strains.

Here, we report for the first time the circulation of dengue virus type 1 (DENV-1) belonging to the lineage IV of genotype V (African American genotype) based on phylogenetic analysis of nucleotide sequences from 10 DENV-1-positive samples obtained in Mexico between 2012 and 2014. Our data revealed that the lineages III and IV of DENV-1 genotype V were found circulating during the same period, probably explaining the rise in the number of cases of severe dengue during that period.

The axolotl (Ambystoma mexicanum) is the vertebrate model system with the highest regeneration capacity. Experimental tools established over the past 100 years have been fundamental to start unraveling the cellular and molecular basis of tissue and limb regeneration. In the absence of a reference genome for the Axolotl, transcriptomic analysis become fundamental to understand the genetic basis of regeneration. Here we present one of the most diverse transcriptomic data sets for Axolotl by profiling coding and non-coding RNAs from diverse tissues. We reconstructed a population of 115,906 putative protein coding mRNAs as full ORFs (including isoforms). We also identified 352 conserved miRNAs and 297 novel putative mature miRNAs. Systematic enrichment analysis of gene expression allowed us to identify tissue-specific protein-coding transcripts. We also found putative novel and conserved microRNAs which potentially target mRNAs which are reported as important disease candidates in heart and liver.

Integrons are non-mobile bacterial genetic elements that carry different cassettes conferring antibiotic resistance. Cassettes can excise or integrate by action of an integron-encoded integrase, enabling bacteria to face environmental challenges. In this work, the functionality and dynamics of two integrons carrying the same cassette arrangement (dfrA12–orfF–aadA2), but located on plasmid or chromosome in two different strains were studied. In order to demonstrate the functionality of the Class 1 integrase, circular cassette integration intermediaries were PCR amplified by PCR using extrachromosomal DNA extracted from bacteria grown in the presence or absence of cassette-encoded antibiotics. Circular aadA2 and dfrA12–orfF–aadA2 cassettes were detected in cultures grown either in the presence or absence of antibiotics in both strains. No dfrA12–orfF circular intermediates could be detected under any culture conditions. These results show that both integrons are functional. However, these elements show different dynamics and functionality since the presence of streptomycin led to detectable gene rearrangements in the variable region only in the strain with the plasmid-born integron. In addition, complete integration products were demonstrated using a receptor molecule carrying an empty integron. In this case, integration products were observed in both strains even in the absence of antibiotics, but they were more evident in the strain with the plasmid-located integron when streptomycin was present in the culture medium. This suggests that integrons in the two strains respond differently to streptomycin even though DNA sequences upstream the intI1 gene, including the lexA boxes of both integrons are identical.