Design, Synthesis and Anticandidal Evaluation of Indazole and Pyrazole Derivatives.

Candidiasis, caused by yeasts of the genus Candida, is the second cause of superficial and mucosal infections and the fourth cause of bloodstream infections. Although some antifungal drugs to treat candidiasis are available, resistant strains to current therapies are emerging. Therefore, the search for new candicidal compounds is certainly a priority. In this regard, a series of indazole and pyrazole derivatives were designed in this work, employing bioisosteric replacement, homologation, and molecular simplification as new anticandidal agents. Compounds were synthesized and evaluated against C. albicans, C. glabrata, and C. tropicalis strains. The series of 3-phenyl-1H-indazole moiety (10a-i) demonstrated to have the best broad anticandidal activity. Particularly, compound 10g, with N,N-diethylcarboxamide substituent, was the most active against C. albicans and both miconazole susceptible and resistant C. glabrata species. Therefore, the 3-phenyl-1H-indazole scaffold represents an opportunity for the development of new anticandidal agents with a new chemotype.

Analysis of inflammatory cytokine expression in the urinary tract of BALB/c mice infected with Proteus (P.) mirabilis and enteroaggregative Escherichia (E.) coli (EAEC) strains.

This study aimed to analyze the proinflammatory cytokine mRNA expression in the urinary tract of BALB/c mice infected with bacterial strains with uropathogenic potential. Groups of four 6-week-old female BALB/c mice were intraurethrally inoculated with 5 × 107 colony-forming units (CFU) of P. mirabilis ATCC29906, EAEC O42, P. mirabilis RTX339, or sterile saline (control group) and then sacrificed at 0, 2, 4, 7, or 10 days post-infection (p.i.). Samples were cultured to determine the CFU/mL in urine or CFU/g in the bladders and kidneys. Cytokine expression (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, -6, and -8) was evaluated in the target organs using real-time PCR and immunohistochemistry; histology was examined with hematoxylin and eosin staining. The results are presented as the means and standard deviations and were compared using one-way ANOVA, with p < 0.05 indicating significant differences. Bacteriuria was not detected in the infected groups; bacterial colonization occurred in the target organs at all time points, but was higher in mice infected with EAEC O42 or P. mirabilis RTX339 at 7 days p.i. The expression of all cytokine mRNAs was seen, but only the levels of the IL-8 protein increased in situ at 7 days p.i. in the P. mirabilis RTX339 and EAEC O42 groups in both organs. Morphological alterations, observed in all of the infected groups, were more prominent in the EAEC O42 and P. mirabilis RTX339 groups. The findings provide insights into the uropathogenicity and inflammatory cytokine expression in the urinary tract of mice infected with three previously untested bacterial strains.

Impact of chronic immobilization stress on parameters of colonic homeostasis in BALB/c mice.

The intestinal epithelium is a monolayer of cells arranged side­by­side and connected by tight junction (TJ) proteins expressed at the apical extreme of the paracellular membrane. This layer prevents stress­induced inflammatory responses, thus helping to maintain gut barrier function and gut homeostasis. The aim of the present study was to evaluate the effects of chronic immobilization stress on the colonic expression of various parameters of homeostasis. A total of two groups of female BALB/c mice (n=6) were included: A stressed group (short­term immobilization for 2 h/day for 4 consecutive days) and an unstressed (control) group. Colon samples were obtained to detect neutrophils and goblet cells by optical microscopy, TJ protein expression (occludin, and claudin ­2, ­4, ­7, ­12 and ­15) by western blotting, mRNA levels of TJ genes and proinflammatory cytokines [tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, ­6 and ­8] by reverse transcription­quantitative PCR, fecal lactoferrin by ELISA and the number of colony­forming units of aerobic bacteria. Compared with goblet cells in control mice, goblet cells were enlarged and reduced in number in stressed mice, whereas neutrophil cellularity was unaltered. Stressed mice exhibited reduced mRNA expression for all evaluated TJ mRNAs, with the exception of claudin­7, which was upregulated. Protein levels of occludin and all claudins (with the exception of claudin­12) were decreased in stressed mice. Fecal lactoferrin, proinflammatory cytokine mRNA levels and aerobic bacterial counts were all increased in the stressed group. These results indicated that immobilization stress induced proinflammatory and potential remodeling effects in the colon by decreasing TJ protein expression. The present study may be a useful reference for therapies aiming to regulate the effects of stress on intestinal inflammatory dysfunction.

Keratinocyte infection by Actinomadura madurae triggers an inflammatory response.

BACKGROUND: Actinomycetoma is a syndrome of the skin characterized by chronic inflammation and lesions with nodular grain-like structures. The most common aetiological agents are Nocardia brasiliensis and Actinomadura madurae. In response to infection with these organisms the body produces an inflammatory immune response in the skin. The aim of the present study was to determine the production of chemokines, pro-inflammatory cytokines, antimicrobial peptides and the expression of Toll-like receptors (TLRs) in keratinocytes infected by A. madurae. METHODS: A cell line of HaCaT keratinocytes was infected with A. madurae at a multiplicity of infection of 20:1 for 2 h and the samples were collected from 2 to 72 h post-infection. Intracellular replication of the bacterium was evaluated by counting of colony-forming units, the TLR expression and antimicrobial peptide production were assayed by confocal microscopy and chemokine and pro-inflammatory cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: Early in the infection, A. madurae was able to achieve intracellular replication in keratinocytes, however, the cells eventually controlled the infection. In response to the infection, keratinocytes overexpressed TLR2 and TLR6, produced high concentrations of cytokines monocyte chemoattractant protein-1, interleukin 8, human ß-defensin-1, human ß-defensin-2 and LL37 and low levels of tumour necrosis factor α. CONCLUSIONS: The human keratinocytes contribute to the inflammatory process in response to A. madurae infection by overexpressing TLRs and producing chemokines, pro-inflammatory cytokines and antimicrobial peptides.

Synthesis and Biological Evaluation of 2H-Indazole Derivatives: Towards Antimicrobial and Anti-Inflammatory Dual Agents.

Indazole is considered a very important scaffold in medicinal chemistry. It is commonly found in compounds with diverse biological activities, e.g., antimicrobial and anti-inflammatory agents. Considering that infectious diseases are associated to an inflammatory response, we designed a set of 2H-indazole derivatives by hybridization of cyclic systems commonly found in antimicrobial and anti-inflammatory compounds. The derivatives were synthesized and tested against selected intestinal and vaginal pathogens, including the protozoa Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis; the bacteria Escherichia coli and Salmonella enterica serovar Typhi; and the yeasts Candida albicans and Candida glabrata. Biological evaluations revealed that synthesized compounds have antiprotozoal activity and, in most cases, are more potent than the reference drug metronidazole, e.g., compound 18 is 12.8 times more active than metronidazole against G. intestinalis. Furthermore, two 2,3-diphenyl-2H-indazole derivatives (18 and 23) showed in vitro growth inhibition against Candida albicans and Candida glabrata. In addition to their antimicrobial activity, the anti-inflammatory potential for selected compounds was evaluated in silico and in vitro against human cyclooxygenase-2 (COX-2). The results showed that compounds 18, 21, 23, and 26 display in vitro inhibitory activity against COX-2, whereas docking calculations suggest a similar binding mode as compared to rofecoxib, the crystallographic reference.

Activation and IL-1ß secretion of human peripheral phagocytes infected with Actinomadura madurae, Nocardia asteroides and Candida albicans.

OBJECTIVE: To evaluate the ability of Actinomadura madurae (A. madurae) and Nocardia asteroides (N. asteroides), using Candida albicans (C. albicans) as prototypic control, to elicit the activation and IL-1ß secretion of blood phagocytic cells from healthy donors. METHODS: Microscopic evaluation of phagocytosis/activation, cell viability and spectrophotometric quantitation of endocytosis/activation, were assessed by using formazan blue test in human blood phagocytes infected with C. albicans, A. madurae or N. asteroides treated with either normal human serum (NHS) or with decomplemented NHS. Interlukin-1ß from culture supernatants of infected polymorphonuclear was tested by ELISA kit assay. RESULTS: Microscopic assay showed that phagocytosis and activation of adherent mononuclear phagocytes were greater with C. albicans followed by A. madurae and then by N. asteroides. Spectrophotometric assay in polymorphonuclear phagocytes infected with NHS-treated pathogens indicated that activation was similarly higher by C. albicans and A. madurae and lower by N. asteroides. Kinetic assays in infected polymorphonuclear cells showed that viability was decreased by C. albicans and N. asteroides or unaffected with A. madurae. Levels of IL-1ß at 8 h of incubation were higher with C. albicans followed by A. madurae whereas lower levels were found with N. asteroides. CONCLUSIONS: The extent of cell-viability and activation as well IL-1ß secretion may be related with the virulence of C. albicans and N. asteroides and other parameters remain to be explored for assessing the virulence of A. madurae.

Antibiotic resistance and multidrug-resistant efflux pumps expression in lactic acid bacteria isolated from pozol, a nonalcoholic Mayan maize fermented beverage.

Pozol is a handcrafted nonalcoholic Mayan beverage produced by the spontaneous fermentation of maize dough by lactic acid bacteria. Lactic acid bacteria (LAB) are carriers of chromosomal encoded multidrug-resistant efflux pumps genes that can be transferred to pathogens and/or confer resistance to compounds released during the fermentation process causing food spoiling. The aim of this study was to evaluate the antibiotic sensibility and the transcriptional expression of ABC-type efflux pumps in LAB isolated from pozol that contributes to multidrug resistance. Analysis of LAB and Staphylococcus (S.) aureus ATCC 29213 and ATCC 6538 control strains to antibiotic susceptibility, minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) to ethidium bromide were based in "standard methods" whereas the ethidium bromide efflux assay was done by fluorometric assay. Transcriptional expression of efflux pumps was analyzed by RT-PCR. LAB showed antibiotic multiresistance profiles, moreover, Lactococcus (L.) lactis and Lactobacillus (L.) plantarum displayed higher ethidium bromide efflux phenotype than S. aureus control strains. Ethidium bromide resistance and ethidium bromide efflux phenotypes were unrelated with the overexpression of lmrD in L. lactics, or the underexpression of lmrA in L. plantarum and norA in S. aureus. These findings suggest that, moreover, the analyzed efflux pumps genes, other unknown redundant mechanisms may underlie the antibiotic resistance and the ethidium bromide efflux phenotype in L. lactis and L. plantarum. Phenotypic and molecular drug multiresistance assessment in LAB may improve a better selection of the fermentation starter cultures used in pozol, and to control the antibiotic resistance widespread and food spoiling for health safety.

Genotypic characterization of methicillin-resistant Staphylococcus aureus strains isolated from the anterior nares and catheter of ambulatory hemodialysis patients in Mexico.

Methicillin-resistant Staphylococcus aureus (MRSA) is the causal agent of multiple nosocomial infections worldwide, including catheter-associated bacteremia in hemodialysis patients. The purposes of this work were to genetically characterize a group of MRSA isolates from catheter-related infections of ambulatory Mexican hemodialysis patients and to determine whether the strains are the same as those carried by the patients in their anterior nares. Sixteen pairs of MRSA isolates from the catheter (cat) and anterior nares (N) of hemodialysis patients were compared using pulsed-field gel electrophoresis (PFGE), PCR detection of adhesion genes and other virulence markers, and an antibiogram. Three pairs of N/cat MRSA isolates (18.7 %) with identical resistograms also showed the same combination of PCR-detected markers and PFGE pattern; one additional pair showed only an identical electrophoretic PFGE pattern. Of the MRSA isolates, 75 % (n = 24) were resistant to ≥ 7 antibiotics, 4 isolates were resistant to 11 antibiotics, and 7 isolates were resistant to the 12 antibiotics tested. The most frequent virulence marker combination found was spa, clfA, clfB, cna, bbp, ebps, map/eap, sdrC, sdrD, sdrE, ica, agr (65.6 %, n = 21). The SCCmec alleles of the 32 MRSA isolates were IV (n = 20), I (n = 7), II (n = 4), and V (n = 1), and no SCCmec type III MRSA was found. The genotypic characterization of the MRSA isolates studied in this work will contribute to a better understanding of the virulence gene makeup of catheter-colonizing S. aureus strains and will help to lower the infection risk in these patients.

Isolation of community-acquired methicillin-resistant Staphylococcus aureus in healthy carriers in a Mexican community.

OBJECTIVES: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) clones are spreading rapidly among the population in many regions worldwide. Little information is available on CA-MRSA in Mexico. The aim of this study was to identify CA-MRSA strains in the nose and throat of healthy people in a Mexican community. METHODS: A total of 131 MRSA strains from the nose and throat obtained from healthy people in Mexico City were characterized. The genes mecA, lukS-PV/lukF-PV, and ACME-arcA were detected by PCR. Staphylococcal cassette chromosome mec (SCCmec), pulsed-field gel electrophoresis (PFGE), and spa typing were performed. RESULTS: Bacteria that had a Panton-Valentine leukocidin (PVL)-positive gene and SCCmec type IV or V were designated as CA-MRSA strains. We found that 21.4% of MRSA strains were CA-MRSA and that the percentage of CA-MRSA strains was similar in the nose and the throat. A great diversity of profiles was found in the strains identified by PFGE pattern and spa typing. Only one strain similar to the USA300 genotype was found; this strain carried the ACME-arcA gene. CONCLUSIONS: CA-MRSA strains were detected in the nose and throat of healthy people. We identified a high level of genetic diversity among CA-MRSA strains in healthy people of Mexico City, which were different from the USA and pandemic clone profiles.

Novel Organotin(IV) Schiff Base Complexes with Histidine Derivatives: Synthesis, Characterization, and Biological Activity.

Five novel tin Schiff base complexes with histidine analogues (derived from the condensation reaction between L-histidine and 3,5-di-tert-butyl-2-hydroxybenzaldehyde) have been synthesized and characterized. Characterization has been completed by IR and high-resolution mass spectroscopy, 1D and 2D solution NMR ((1)H, (13)C and (119)Sn), as well as solid state (119)Sn NMR. The spectroscopic evidence shows two types of structures: a trigonal bipyramidal stereochemistry with the tin atom coordinated to five donating atoms (two oxygen atoms, one nitrogen atom, and two carbon atoms belonging to the alkyl moieties), where one molecule of ligand is coordinated in a three dentate fashion. The second structure is spectroscopically described as a tetrahedral tin complex with four donating atoms (one oxygen atom coordinated to the metal and three carbon atoms belonging to the alkyl or aryl substituents), with one molecule of ligand attached. The antimicrobial activity of the tin compounds has been tested against the growth of bacteria in vitro to assess their bactericidal properties. While pentacoordinated compounds 1, 2, and 3 are described as moderate effective to noneffective drugs against both Gram-positive and Gram-negative bacteria, tetracoordinated tin(IV) compounds 4 and 5 are considered as moderate effective and most effective compounds, respectively, against the methicillin-resistant Staphylococcus aureus strains (Gram-positive).

Frequency and expression of ALS and HWP1 genotypes in Candida albicans strains isolated from Mexican patients suffering from vaginal candidosis.

To detect the frequency and expression of eight ALS (agglutinin-like sequence) genes and the HWP1 genotype in a group of Candida albicans strains isolated from Mexican women suffering from vaginal candidosis. A group of 264 women (age 15-57 years) with vaginal infections were evaluated. C. albicans was identified by PCR amplification of the rRNA internal transcribed spacer regions ITS1 and ITS2. The ALS and HWP1 genes were identified by conventional PCR, and their expression levels were determined by real-time PCR after growing C. albicans strains in reconstituted human vaginal epithelium (RHVE). C. albicans was identified in 50 women (18.9%). The genotypic frequencies were ALS1 100%, ALS2 60%, ALS3 36%, ALS4 54%, ALS5 70%, ALS6 56%, ALS7 64%, ALS9 66% and HWP1 92%. The most frequently expressed genes in the strains harbouring all of the genes were ALS4 (100%), ALS1 (87.5%), ALS2 (87.5%), ALS3 (87.5%), ALS5 (87.5%), ALS7 (87.5%) and HWP1 (75.0%). Nineteen per cent of the vaginal infections were caused by C. albicans, and a high proportion of the strains carried genes encoding proteins involved in adhesion to epithelia. The ALS and HWP1 genes were expressed in RHVE, suggesting that the Als and Hwp1 proteins play an important role in the pathogenesis of the infection.

Biochemical characterization of a bacteriocin-like inhibitory substance produced by Enterococcus faecium MXVK29, isolated from Mexican traditional sausage.

BACKGROUND: Enterococci are lactic acid bacteria that can produce bacteriocins, which may offer an additional hurdle to control the growth of food-borne pathogens; moreover, these bacteriocins may have great potential as natural biopreservatives. The aim of this work was to characterize a bacteriocin-like inhibitory substance (BLIS) with antilisterial activity produced by an enterococcal strain. RESULTS: The bacteriogenic strain was isolated from Mexican fermented sausages and identified as Enterococcus faecium with 99% sequence similarity. Maximal activity was detected at 16 h, where bacterial growth was in middle of the stationary phase. The producer strain was not inhibited by its own antimicrobial peptide. BLIS showed a strong anti-Listeria activity and was inactivated by proteinase K. Heating (121 °C for 15 min) induced some inactivation, but thermotolerance was higher at acid pH values. The yield obtained with a pH-mediated purification process was 32.7%, showing a band with an estimated molecular weight of 3.5 kDa. Automated N-terminal Edman degradation showed the following sequence: YYGNGVTCGSHHCSVD. CONCLUSION: Biochemical characteristics of BLIS produced by E. faecium MXVK29 suggested that it belongs to Class IIa of the Klaenhammer classification and could be considered as a natural food preservative, although further studies need to be performed.

Characterization and persistence of Staphylococcus aureus strains isolated from the anterior nares and throats of healthy carriers in a Mexican community.

Healthy carriers of Staphylococcus aureus strains have an important role in the dissemination of this bacterium. To investigate the presence of S. aureus in the throat and anterior nares, samples from 1,243 healthy volunteers in a Mexican community were examined. The percentage of healthy carriers was 59.8%. Results showed that colonization of the throat occurred more frequently than that of the nares (46.5% versus 37.1%, P<0.0001). Of the S. aureus carriers, 22.2% were exclusive nasal carriers and 38% were exclusive throat carriers. A total of 1,039 strains were isolated; 12.6% were shown to be methicillin-resistant S. aureus (MRSA). Of MRSA strains, 32.1% were isolated from exclusive throat carriers. Most of the strains isolated from the anterior nares and throat of the same carriers were the same or related; however, some were different. Pulsed-field gel electrophoresis (PFGE) pattern analysis of the MRSA strains isolated from the exclusive nasal carriers or exclusive throat carriers showed that they belong to different clusters. A 6-year prospective study was performed to investigate the persistence of S. aureus in the throat. Results showed that 13% of subjects were persistent carriers. Most of them were colonized with the same clone of S. aureus throughout the time of the study, and just three had different clones. Antimicrobial susceptibility testing showed that 91.1% of the strains were penicillin resistant. The presence of mecA and nucA genes (in order to confirm methicillin resistance) and of thermostable nuclease of S. aureus was examined. This study showed that some strains of S. aureus regularly colonized the throats of healthy people and could persist for years.

Citometría de fuujo: vínculo entre la investigación básica y la aplicación clínica

La citometría de flujo es un método analítico que permite la medición rápida de ciertas características físicas y químicas de células o partículas suspendidas en líquido que producen una señal de forma individual al interferir con una fuente de luz. Una de las características analíticas más importantes de los citómetros de flujo es su capacidad de medir múltiples parámetros celulares, como el tamaño, forma y complejidad y, por supuesto, cualquier componente celular o función que pueda ser marcada con un fluorocromo. Las aplicaciones más relevantes de la citometría de flujo en la práctica médica se relacionan con la hematología e inmunología clínicas, midiendo parámetros como número y clasificación de células sanguíneas. Esta técnica es empleada también en el conteo de subpoblaciones de linfocitos en pacientes con el virus de la inmunodeficiencia humana, así como la caracterización de leucemias agudas y síndromes linfoproliferativos crónicos, entre otros padecimientos. En los últimos 20 años, el análisis de enfermedades pulmonares de origen inmunológico por citometría de flujo ha jugado un papel importante en el entendimiento y diagnóstico de enfermedades como sarcoidosis, neumonía eosinofílica o neumonitis por hipersensibilidad. Las aplicaciones de la citometría de flujo son numerosas, lo cual ha permitido el empleo de estos instrumentos de manera amplia en los campos, tanto de la investigación biológica como médica. Esta revisión brinda un panorama general de los principios básicos de la citometría de flujo y la muestra como una herramienta reproducible y aplicable a una gran variedad de campos médicos, así como su empleo en el campo de las enfermedades pulmonares.

Flow cytometry is an analytical method that allows the rapid measurement of certain physical and chemical characteristics of cells or particles suspended in liquid and produce signals when they pass individually through a beam of light. An important analytical feature of flow cytometers is their ability to measure multiple cellular parameters such as cell size, shape and internal complexity and, of course any cell component or function that can be detected by a fluorescent dye. The most prominent uses of flow cytometry in medical practice are in the related fields of laboratory hematology and clinical immunology, for a variety of tasks involving blood cell counting and classification. This technique is also used for counting lymphocyte subpopulations in patients with HIV, characterization of acute leukemias and chronic lymphomas between other diseases. Over the last 20 years, analysis of immunologica lung diseases by flow cytometry has played a major role in the understanding and as tool of diagnosis, such as sarcoidosis, eosinophilic pneumonia or hypersensitivity pneumonitis. So the applications of flow cytometry are numerous, and this has lead to the widespread use of this instruments in biological research and medical fields. Overall this review shows a brief overview of basic principles of and shows this as a reproducible tool applicable to a wide range of medical approaches as well as its use in lung diseases field.

Antibody response in mice immunized by mucosal routes with formalin-inactivated enteropathogenic Escherichia coli (EPEC) strains.

Secretory and systemic antibody response in mice against enteropathogenic Escherichia coli (EPEC) was evaluated. Groups of mice were immunized with formalin inactivated EPEC 0127:H6 strain by intranasal, peroral, intragastric and intrarectal route, with and without cholera toxin (CT) used as mucosal adjuvant. Mice immunized subcutaneously and a non treated control group were included. Other groups of mice were immunized intranasally with different EPEC strains and a non pathogenic E. coli K12 strain. Antibody response tested by ELISA assay showed that specific anti EPEC 0127:H6 antibodies were induced in serum by intranasal, subcutaneous and intragastric routes. A strong increase of antibody response against EPEC 0127:H6 strain was observed in saliva after intranasal delivery, while a lower response was detected by peroral and intrarectal immunization. Only the intranasal route increased IgA anti EPEC 0127:H6 antibody titers in feces. Specific and cross reactive antibodies to EPEC 0127:H6 were seen in mice immunized intranasally with different EPEC strains. Some control mice showed a background of anti EPEC 0127:H6 antibodies in feces. CT had a negative effect as adjuvant. We showed that nasal mucosa rendered the strongest antibody response in serum and secretions. These results might contribute to optimize the protective effect of enteric vaccines against infections associated to EPEC.