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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.

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.

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.

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.

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.

Resumen Antecedentes Los hongos patogenos han desarrollado estrategias que involucran la expresion de genes que les ayudan a permanecer y multiplicarse en el huesped. La ausencia de las moleculas codificadas por estos genes podria interferir en el crecimiento y muerte de estos hongos. En el pasado, se reporto un gen (Hcp100) que codifica para una proteina coactivadora del hongo Histoplasma capsulatum , el cual se sobreexpresa despues de 1 h de contacto entre las levaduras del hongo y los macrofagos murinos. El producto de este gen, una proteina de 100 kDa (Hcp100) de H. capsulatum , es probablemente una proteina regulatoria involucrada en los procesos necesarios de adaptacion del hongo para la supervivencia en las condiciones hostiles intracelulares de los macrofagos. Un fragmento de 210 pb del marcador Hcp100 ha revelado ser una excelente herramienta para la deteccion molecular de H. capsulatum en muestras clinicas. El potencial uso de este gen como blanco terapeutico en Plasmodium falciparum ha sido explorado a traves de la inhibicion del gen y de la proteina p100 del parasito, logrando bloquear su crecimiento. Metodos Tomando como base los antecedentes mencionados, la Hcp100 podria tener un papel clave en el desarrollo y mantenimiento de las levaduras de H. capsulatum dentro de los macrofagos. Resultados y conclusiones Estudiar la probable funcion de la Hcp100 en la fase de levadura de este patogeno fungico es relevante para entender su actividad y poder proponerla como un blanco terapeutico para el tratamiento de la histoplasmosis.

Plasmids belonging to the IncU incompatibility group are mobile genetic elements isolated frequently from Aeromonas spp. These plasmids share structural and functional characteristics and often carry Class-1 integrons bearing antibiotic resistance genes. In this work the ability of two IncU plasmids, pAr-32 and pRAS1 to establish in different A. hydrophila strains after conjugal transfer was studied. In vitro transfer frequencies on solid surface ranged from 10(-1) to 10(-6) for pAr-32 and from 10(-3) to 10(-5) for pRAS1. While carrying out these experiments we detected four strains unable to acquire plasmid pRAS1, indicating that the genetic background of recipients affects the establishment of the plasmid. We explored the possible reasons why these strains failed to yield transconjugants after mating experiments using A. salmonicida 718 as a donor. Factors included donor cell recognition, incompatibility, surface exclusion and restriction of incoming DNA. We found that none of these factors could explain the refractivity of non-receptive A. hydrophila strains to yield transconjugants. Although we do not know the reasons of this refractivity, we may speculate that these isolates lack a product necessary to replicate or stabilize plasmid pRAS1. Alternatively, these strains could contain a product that impedes plasmid establishment.

BACKGROUND:Theimpactofmixturesofchloro-triazinicherbicides,suchasatrazineandsimazine,onaquaticecosystemsisof environmental concern. To study their biodegradation under various operational conditions, a binary community comprising StenotrophomonasmaltophiliaandArthrobacter sp. attached to the porous support of a packed bed reactor, was evaluated. RESULTS: The genetic analysis of the two atrazine-degrading strains revealed that genes atzA, atzB, atzC are present in both bacteria, but only S. maltophilia possess atzD. Thus, by cultivating Arthrobacter sp. on these herbicides, cyanuric acid accumulation was observed. When the binary community was cultivated in the biofilm reactor, at all the loading rates probed, both herbicides were entirely removed. However, complete biodegradation of cyanuric acid was not achieved. CONCLUSIONS: Even with a two-stage reactor, cyanuric acid was only partially removed. This fact could be attributed to the absence, in the second stage, of an easily degradable energy source, required by S.maltophiliafor the uptake and cometabolic degradation of the recalcitrant heterocyclic ring. Responding to differences in nutritional conditions prevailing at each reactor stage, local differences in species’ predominance were clearlydetected by microbiological and molecular biology methods. c � 2010 Society of Chemical Industry

We determined the presence of class 1 integrons related to the acquisition of resistance to antimicrobials in Aeromonas spp. isolated from individuals with diarrhea. Species were identified as A. caviae, A. hydrophila, A. veronii and A. media using PCR-RFLP of the 16S rDNA. Selected isolates were further characterized by ERIC-PCR. Resistance to chloramphenicol, aztreonam, tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid and streptomycin, among others, was determined using the Kirby-Bauer method. Integrons were detected by PCR amplification of the 5' conserved, variable, and 3' conserved regions. Sequencing of the variable regions revealed class 1 integrons with cassettes encoding resistance to trimethoprim (dfrA12, dfrA15, dfrB4), streptomycin/spectinomycin (aadA2, aadA1), oxacillin (oxa2) and chloramphenicol (catB3, cmlA4). Others had an open reading frame (orfD) or no insert at all. To our knowledge, this is the first description of the occurrence of genes cmlA4 and dfrA15 in Aeromonas class 1 integrons. Not all the integron-linked cassettes conferred their associated resistances, which suggests the inactivity of some cassettes. Most integrons were chromosomally located. The presence of class 1 integrons similar to those found in a wide variety of bacterial genera from different origins, including environmental and fish-borne Aeromonas, confirms the stability and horizontal transfer of these genetic elements.

Using a successive transfer method on mineral salt medium containing simazine, a microbial community enriched with microorganisms able to grow on simazine was obtained. Afterwards, using a continuous enrichment culture procedure, a bacterial community able to degrade simazine from an herbicide formulation was isolated from a chemostat. The continuous selector, fed with a mineral salt medium containing simazine and adjuvants present in the commercial herbicide formulation, was maintained in operation for 42 days. Following the lapse of this time, the cell count increased from 5 × 105 to 3 × 108 CFU mL−1, and the simazine removal efficiency reached 96%. The chemostat’s bacterial diversity was periodically evaluated by extracting the culture’s bacterial DNA, amplifying their 16S rDNA fragments and analyzing them by thermal gradient gel electrophoresis. Finally, a stable bacterial consortium able to degrade simazine was selected. By PCR amplification, sequencing of bacterial 16S rDNA amplicons, and comparison with known sequences of 16S rDNA from the NCBI GenBank, eight bacterial strains were identified. The genera, Ochrobactrum, Mycobacterium, Cellulomonas, Arthrobacter, Microbacterium, Rhizobium and Pseudomonas have been reported as common degraders of triazinic herbicides. On the contrary, we were unable to find reports about the ability of the genus Pseudonocardia to degrade triazinic compounds. The selected bacterial community was attached to a porous support in a concurrently aerated four-stage packed-bed reactor fed with the herbicide. Highest overall simazine removal efficiencies η SZ were obtained at overall dilution rates D below 0.284 h−1. However, the multistage packed bed reactor could be operated at dilution rates as high as D = 3.58 h−1 with overall simazine removal volumetric rates R v,SZ = 19.6 mg L−1 h−1, and overall simazine removal specific rates R X,SZ = 13.48 mg (mg cell protein)−1 h−1. Finally, the consortium’s ability to degrade 2-chloro-4,6-diamino-1,3,5-triazine (CAAT), cyanuric acid and the herbicide atrazine, pure or mixed with simazine, was evaluated in fed batch processes.

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of β-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.Key words: translation initiation, in-frame gene cloning, expression vectors.

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.

Mercury-resistant Aeromonas strains isolated from diarrhea were studied. Resistance occurs via mercuric ion reduction but merA and merR genes were only detected in some strains using PCR and dot hybridization. Results indicate a high variability in mer operons in Aeromonas. To our knowledge, this is the first report of mercury-resistant clinical Aeromonas strains.

In spite of a well-documented ability of Samonella enterica Typhi strains to receive R factors from Escherichia coli and other enterobacteria, epidemiological data show that Typhi is a rather poor host of antibiotic-resistance genes and in fact, of plasmids, suggesting that most of the plasmids naturally acquired by Typhi strains become unstable and eventually segregate. We have previously reported evidence that each of three plasmids conjugatively transferred to S. enterica Typhi experienced deletion-mediated loss of a resistance determinant before plasmid segregation occurred. We now report that in Typhi strains containing these unstable plasmids a superhelical DNA species of lower mobility is detected, probably representing plasmid dimer structures. Plasmid deletion is a RecA-dependent process since it is not detected in derivatives of a recA1 S. enterica Typhi strain containing the corresponding plasmids, and in such strains we were unable to detect either the low-mobility species. We propose that the deletable segments contain key information for plasmid stability in S. enterica Typhi, possibly a multimer resolution system.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon-tagged A. hydrophila strains using mini Tn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10(-3) and 10(-6) and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 x 10(4) CFU mL(-1). Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.

A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recongnized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host–parasite relationship of histoplasmosis.

Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries.

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the sacroiliac joints and vertebral column of unknown aetiology, but strongly related to the presence of the HLA-B27 antigen. The participation of bacterial infections as triggering factors have also been suggested. We have associated the 60 kDa heat shock protein of Klebsiella pneumoniae (HSP60Kp) with AS since we have previously demonstrated that most of the patients have IgG antibodies and active T cells that recognize preferentially this protein, but we have not yet identified the epitopes involved in the recognition. In order to know the amino acid sequence of HSP60Kp, and to be able to analyse in the future the relevant epitopes, we amplified by PCR and cloned the gene coding for this protein into the Sma I site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniques. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this sequence was compared to the antigenic analysis of the reported sequences of Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a software to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) residues 117-125 of HSP60Kp showed a much higher affinity for HLA-B27 than the similar nonapeptide of E. coli GroEL and Y. enterocolitica HSP60. The only difference between the three peptides was in position nine. This finding could explain the association of AS only with the HSP60 of Klebsiella pneumoniae . On the other hand, hydrophilicity analysis, which indicates B cell epitopes, showed three similar strongly antigenic regions in the three proteins.