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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.

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.

Resumen Introducción: Los alimentos de origen animal frecuentemente están implicados en brotes de salmonelosis. Objetivo: Evaluar la frecuencia de Salmonella enterica en carnes molidas de pollo, res y cerdo (un total de 2.592 muestras) obtenidas de mercados sobre ruedas y supermercados de la Delegación Iztapalapa en la Ciudad de México, determinar la susceptibilidad antimicrobiana y efectuar ensayos de adherencia en las cepas aisladas. Métodos: El aislamiento de S. enterica se hizo de acuerdo a la BAM-FDA, la susceptibilidad antimicrobiana de acuerdo con CLSI y el ensayo de adherencia en células HEp-2 conforme a Baffone y cols., 2001. Resultados: Salmonella enterica fue aislada en 511 del total de muestras analizadas (19,7%), de las cuales 244 (47,7%), 152 (29,7%) y 115 (22,5%) correspondieron a carne molida de pollo, res y cerdo, respectivamente. La mayor frecuencia de resistencia de S. enterica a antimicrobianos fue a ampicilina y cloranfenicol en pollo, perfloxacina y ampicilina en res y carbenicilina, ampicilina, cloranfenicol, cefotaxima y perfloxacina en cerdo. Noventa por ciento de las cepas mostraron un patrón de adherencia agregativo. Conclusión: La frecuencia de S. enterica en productos cárnicos es alta, por lo que es importante la adecuada cocción de la carne para disminuir el riesgo de una salmonelosis.

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.

Background: Cronobacter sakazakii is an opportunistic foodborne pathogen associated with necrotizing enterocolitis, bacteraemia, and meningitis in infants. A comparative proteomic study of C . sakazakii ATCC BAA-894 (CS WT) and isogenic mutant of flagella were performed; including the ability of both strains to adhere and invade to N1E-115 cells. Results: To achieve this goal, a non-motile C. sakazakii ATCC BAA-894 fliF ::Tn5 (CS fliF ::Tn5) strain was generated using an EZ-Tn5Tnp Transposome kit. Analysis of differential protein expression showed that 81.49% (361/443) of the proteins were identified in both strains, 8.35% (37/443) were exclusively expressed in the CS WT strain and 10.16% (45/443) in the CS fliF ::Tn5 strain. The main exclusive proteins from the CS WT strain were classified into the following subcategories: “cell motility” and “signal transduction mechanisms”. In contrast, the exclusive proteins from the CS fliF ::Tn5 strain were classified into the following subcategories: “intracellular trafficking, secretion, and vesicular transport”, “replication, recombination, and repair”, “nucleotide transport and metabolism”, “carbohydrate transport and metabolism”, “coenzyme transport and metabolism”, and “lipid transport and metabolism”. Expression of the Cpa protein was shared by both strains but was more abundant in the CS WT strain than in the CS fliF ::Tn5 strain. Significant increases (p=0.0001) in adherence to N1E-115 cells were observed for the non-motile CS fliF ::Tn5 strain, with 31.3×10 6 CFU/mL, relative to the CS WT strain, with 14.5×10 6 CFU/mL. Additionally, for infection of N1E-115 cells, the CS WT strain showed a 0.17% invasion frequency, which was significantly increased (p=0.01) compared to that of the non-motile CS fliF ::Tn5 strain. Conclusion : The proteins involved in motility were mainly identified by proteomic analysis in CS WT strain when compare to CS fliF ::Tn5 strain. Our data showed that flagella are required to promote invasion to N1E-115 cells and absence of flagella significantly increases the adherence to N1E-115 cells when compare to CS WT strain.

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 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.

Food of animal origin is often involved in salmonellosis outbreaks.To evaluate the frequency of Salmonella enterica in chicken, beef and pork ground meat (a total of 2,592 samples) obtained from travelling markets and supermarkets at the Iztapalapa area of Mexico City, in order to determine the antimicrobial susceptibility and adherence capacity of isolated strains.Isolation of S. enterica was carried out according to the BAM-FDA, the microbial susceptibility according with CLSI and adherence assay on HEp-2 cell line according with Baffone et al., 2001.S. enterica was isolated from 511 of all the analyzed samples (19.7%), from which 244 (47.7%), 152 (29.7%) and 115 (22.5%) corresponded to chicken, beef and pork ground meat, respectively. The highest frequency of resistance of S. enterica to antimicrobials was to ampicillin and chloramphenicol in chicken, perfloxacin and ampicillin in beef and carbenicillin, ampicillin, chloramphenicol, cefotaxime and perfloxacin in pork. Ninety percent of the strains showed an aggregative adherence pattern on HEp-2 cells.The frequency of S. enterica on meat products is high, which is the reason why a proper cooking of these ground meats is important in order to reduce the risk of acquiring salmonellosis.

Vibrio alginolyticus has acquired increasing importance because this microorganism may be pathogenic to aquatic animals and humans. It has been reported that some V. alginolyticus strains carry virulence genes derived from pathogenic V. cholerae and V. parahaemolyticus strains. In this work V. alginolyticus was isolated from oyster samples acquired from a food-market in Mexico City. Thirty isolates were identified as V. alginolitycus. Strains showed β-haemolysis and proteolytic activity and produced a capsule. Strains displayed swimming and swarming motility and 93.3% of them produced siderophores. Several genes encoding virulence factors were detected using PCR amplification. These included proA, wza, vopD, vopB, hcp, vasH and vgrG genes, which were present in all strains. Other genes had a variable representation: tdh (86.6%), lafA (96.6%), pvsA (62%) and pvuA (16%). The trh gene could not be amplified from any of the strains. The antimicrobial resistance profile revealed that more than 90% of the strains were resistant to beta-lactams antibiotics, 60% to cephalotin, 45% to amikacin, 16% to cephotaxime, and 10% to pefloxacin, while 100% were susceptible to ceftriaxone. The V. alginolyticus strains isolated from oysters showed multiple resistance to antibiotics and several virulence factors described in well-characterized pathogenic vibrios. [Int Microbiol 19(4):191-198 (2016)].

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.

A series of selected 2-substituted imidazolines were synthesized in moderate to excellent yields by a modification of protocols reported in the literature. They were evaluated as potential non-classical bioisosteres of AHL with the aim of counteracting bacterial pathogenicity. Imidazolines 18a, 18e and 18f at various concentrations reduced the violacein production by Chromobacterium violaceum, suggesting an anti-quorum sensing profile against Gram-negative bacteria. Imidazoline 18b did not affect the production of violacein, but had a bacteriostatic effect at 100 µM and a bactericidal effect at 1 mM. Imidazoline 18a bearing a hexyl phenoxy moiety was the most active compound of the series, rendering a 72% inhibitory effect of quorum sensing at 100 µM. Imidazoline 18f bearing a phenyl nonamide substituent presented an inhibitory effect on quorum sensing at a very low concentration (1 nM), with a reduction percentage of 28%. This compound showed an irregular performance, decreasing inhibition at concentrations higher than 10 µM, until reaching 100 µM, at which concentration it increased the inhibitory effect with a 49% reduction percentage. When evaluated on Serratia marcescens, compound 18f inhibited the production of prodigiosin by 40% at 100 μM.

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.

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.

Fungal pathogens have developed strategies, involving genes expression that favors their persistence and multiplication in the host. The absence of molecules encoded by these genes could interfere with the growth and death of these fungi. In the past, a coactivator protein coding gene (Hcp100) of the fungus Histoplasma capsulatum was reported, which is overexpressed after 1h of contact between fungal yeast-cells and murine macrophages. The product of this gene, a protein of 100 kDa (Hcp100) of H. capsulatum, is probably a regulatory protein involved in the processes required for fungal adaptation and its survival in the intracellular hostile conditions of the macrophages. A 210-bp fragment of the Hcp100 marker has proved to be an excellent tool for H. capsulatum molecular detection in clinical samples. The potential use of this gene as a therapeutic target in Plasmodium falciparum has been explored through the inhibition of both, the gene and the protein p100 of the parasite, by blocking its growth.Based on the above mentioned antecedents, we believe that the Hcp100 has an important role in the development and maintenance of the H. capsulatum yeast cells within macrophages.To study the probable function of Hcp100 in the yeast-phase of this fungal pathogen is relevant to understand its activity and to propose it as a therapeutic target for histoplasmosis treatment.

To assess the effect of amphotericin B and caspofungin, as well as their combinations in the therapy of experimental disseminated histoplasmosis.BALB/c mice were intraperitoneally infected with four different strains of Histoplasma capsulatum and given to antifungal treatments. The response to intraperitoneal therapy with amphotericin B (0.5, 1.0, and 2.0 mg/kg of body weight) or caspofungin (10 mg/kg of body weight) and their combinations, was evaluated by the quantification of yeast colony-forming units (CFU) per gram of spleen or lung, from each animal. Additionally, the pathogen was monitored histopathologically in the excised organs. Data were analyzed with the Kruskall-Wallis and Tukey tests.Caspofungin was more effective than amphotericin B in reducing the CFU/ g. A synergistic effect was observed when caspofungin (10 mg/ kg) was combined with amphotericin B (0.5 or 1.0 mg/kg). Significant differences in CFU values, H = 119.78 (P = 0.00001), were found among the treatment groups. However, statistical analyses did not reveal significant differences, H = 2.837 (P = 0.428), in the therapeutic responses with the four H. capsulatum strains tested.Combined therapy with amphotericin B and caspofungin could represent an alternative treatment to be explored in severe human histoplasmosis.

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.

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Background Breast cancer is one of the most frequent causes of death in Mexican women over 35 years of age. At molecular level, changes in many genetic networks have been reported as associated with this neoplasia. To analyze these changes, we determined gene expression profiles of tumors from Mexican women with breast cancer at different stages and compared these with those of normal breast tissue samples. Methods 32P-radiolabeled cDNA was synthesized by reverse transcription of mRNA from fresh sporadic breast tumor biopsies, as well as normal breast tissue. cDNA probes were hybridized to microarrays and expression levels registered using a phosphorimager. Expression levels of some genes were validated by real time RT-PCR and immunohistochemical assays. Results We identified two subgroups of tumors according to their expression profiles, probably related with cancer progression. Ten genes, unexpressed in normal tissue, were turned on in some tumors. We found consistent high expression of Bik gene in 14/15 tumors with predominant cytoplasmic distribution. Conclusion Recently, the product of the Bik gene has been associated with tumoral reversion in different neoplasic cell lines, and was proposed as therapy to induce apoptosis in cancers, including breast tumors. Even though a relationship among genes, for example those from a particular pathway, can be observed through microarrays, this relationship might not be sufficient to assign a definitive role to Bik in development and progression of the neoplasia. The findings herein reported deserve further investigation.

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.

The structural gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium, has been cloned in Escherichia coli. Expression of the C-protein gene in E. coli requires an external transcription promoter and prevention of termination of transcription prior to transcription of all or part of the sequence coding for the C protein. The gene for the C protein is within a 5-kilobase DNA fragment, but this fragment does not code for either of the other two major low molecular weight spore proteins, suggesting that the structural genes for these proteins are not tightly linked.

Purpose: HLA class II, p-36 protein, heat shock protein and retinal antigens have been associated with pars planitis (PP), but their participation in the development of the disease are unknown. A search for new molecules related to PP is necessary. This work focused on the identification of peptides recognized by PP patient sera using the phage display method. Methods: Sera of PP patients were used to isolate peptides fused to M13-phage pIII protein. The response of PP and healthy sera to peptides was determined by ELISA. PCR amplification and sequencing of peptide-encoding fragments from clones with high recognition by PP sera were used to characterize displayed peptides. Results: One hundred clones were randomly selected from a phage display library after three panning rounds using serum proteins from a PP patient. The immunologic response level of 100 clones selected were determined with a major number of patients, it was found that one clone was recognized stronger in PP patients sera than in healthy sera (PP vs. healthy; P