New pyridine-based chalcones and pyrazolines with anticancer, antibacterial, and antiplasmodial activities.

New pyridine-based chalcones 4a-h and pyrazolines 5a-h (N-acetyl), 6a-h (N-phenyl), and 7a-h (N-4-chlorophenyl) were synthesized and evaluated by the National Cancer Institute (NCI) against 60 different human cancer cell lines. Pyrazolines 6a, 6c-h, and 7a-h satisfied the pre-determined threshold inhibition criteria, obtaining that compounds 6c and 6f exhibited high antiproliferative activity, reaching submicromolar GI50 values from 0.38 to 0.45 µM, respectively. Moreover, compound 7g (4-CH3) exhibited the highest cytostatic activity of these series against different cancer cell lines from leukemia, nonsmall cell lung, colon, ovarian, renal, and prostate cancer, with LC50 values ranging from 5.41 to 8.35 µM, showing better cytotoxic activity than doxorubicin. Furthermore, the compounds were tested for antibacterial and antiplasmodial activities. Chalcone 4c was the most active with minimal inhibitory concentration (MIC) = 2 µg/mL against methicillin-resistant Staphylococcus aureus (MRSA), while the pyrazoline 6h showed a MIC = 8 µg/mL against Neisseria gonorrhoeae. For anti-Plasmodium falciparum activity, the chalcones display higher activity with EC50 values ranging from 10.26 to 10.94 µg/mL. Docking studies were conducted against relevant proteins from P. falciparum, exhibiting the minimum binding energy with plasmepsin II. In vivo toxicity assay in Galleria mellonella suggests that most compounds are low or nontoxic.

In silico prediction and in vitro assessment of novel heterocyclics with antimalarial activity.

The development of new antimalarials is paramount to keep the goals on reduction of malaria cases in endemic regions. The search for quality hits has been challenging as many inhibitory molecules may not progress to the next development stage. The aim of this work was to screen an in-house library of heterocyclic compounds (HCUV) for antimalarial activity combining computational predictions and phenotypic techniques to find quality hits. The physicochemical determinants, pharmacokinetic properties (ADME), and drug-likeness of HCUV were evaluated in silico, and compounds were selected for structure-based virtual screening and in vitro analysis. Seven Plasmodium target proteins were selected from the DrugBank Database, and ligands and receptors were processed using UCSF Chimera and Open Babel before being subjected to docking using Autodock Vina and Autodock 4. Growth inhibition of P. falciparum (3D7) cultures was tested by SYBR Green assays, and toxicity was assessed using hemolytic activity tests and the Galleria mellonella in vivo model. From a total of 792 compounds, 341 with good ADME properties, drug-likeness, and no interference structures were subjected to in vitro analysis. Eight compounds showed IC50 ranging from 0.175 to 0.990 µM, and active compounds included pyridyl-diaminopyrimido-diazepines, pyridyl-N-acetyl- and pyridyl-N-phenyl-pyrazoline derivatives. The most potent compound (UV802, IC50 0.178 µM) showed no toxicophoric and was predicted to interact with P. falciparum 1-cysperoxidredoxin (PfPrx1). For the remaining 7 hits (IC50 < 1 µM), 3 showed in silico binding to PfPrx1, one was predicted to bind the haloacid dehalogenase-like hydrolase and plasmepsin II, and one interacted with the plasmodial heat shock protein 90.

Molecular diagnosis of intestinal protozoa in young adults and their pets in Colombia, South America.

Intestinal parasitic infections have been considered a relevant public health problem due to the increased incidence worldwide. In developing countries, diarrhea and gastrointestinal symptoms cause impaired work capacity in adults and delayed rate growth in children. Enteric infections of unknown etiology can often lead to misdiagnosis, increased transmission, and morbidity. The aim of this study was to determine the prevalence of intestinal parasites in a young adult population and their pets. Stool samples from 139 university students and 44 companion animals were subjected to microscopy diagnosis using wet mounts, concentration by zinc sulphate flotation and staining techniques (Kinyoun and trichrome stain). Molecular diagnosis of protozoa was also performed by conventional PCR. The mean age was 24 years, 54% individuals were female, 46% were men, and 66% had at least one pet. The overall prevalence for at least one parasite was 74.8% and the rate of polyparasitism was 37.5%. Eighty-three patients (59.7%) were positive for Blastocystis spp., followed by Cryptosporidium spp. 24.5%, Endolimax nana 13.6%, Entamoeba dispar/E. moshkovskii 7.8% and Giardia intestinalis 1.4%. Molecular diagnosis substantially improved Cryptosporidium spp. and Blastocystis spp. detection and allowed to distinguish E. histolytica from commensals in the Entamoeba complex. Student's pets were also examined for parasitism. Samples from 27 dogs, 15 cats, one rabbit and one hen were analyzed, and parasites were detected in 30 (68.2%) as follows: Cryptosporidium spp. (24) Giardia spp. (4), hookworm (3), Endolimax nana (2) and Toxoplasma gondii (1). Overall, university students showed high prevalence of parasitism and polyparasitism suggesting exposure to parasite infected animals and contaminated environments. Cryptosporidium spp. was the predominant pathogen in human and domestic animals, and it was only detected by PCR, pointing out the need for sensitive tests in diagnosis and surveillance. Control strategies to prevent the effects of parasitic infections in young population should consider pets as reservoirs and transmission source.

Anticancer activity of pyrimidodiazepines based on 2-chloro-4-anilinoquinazoline: synthesis, DNA binding and molecular docking.

Multidrug resistance to chemotherapy is a critical health problem associated with mutation of the therapeutic target. Therefore, the development of anticancer agents remains a challenge to overcome cancer cell resistance. Herein, a new series of quinazoline-based pyrimidodiazepines 16a-g were synthesized by the cyclocondensation reaction of 2-chloro-4-anilinoquinazoline-chalcones 14a-g with 2,4,5,6-tetraaminopyrimidine. All quinazoline derivatives 14a-g and 16a-g were selected by the U.S. National Cancer Institute (NCI) for testing their anticancer activity against 60 cancer cell lines of different panels of human tumors. Among the tested compounds, quinazoline-chalcone 14g displayed high antiproliferative activity with GI50 values between 0.622-1.81 µM against K-562 (leukemia), RPMI-8226 (leukemia), HCT-116 (colon cancer) LOX IMVI (melanoma), and MCF7 (breast cancer) cancer cell lines. Additionally, the pyrimidodiazepines 16a and 16c exhibited high cytostatic (TGI) and cytotoxic activity (LC50), where 16c showed high cytotoxic activity, which was 10.0-fold higher than the standard anticancer agent adriamycin/doxorubicin against ten cancer cell lines. COMPARE analysis revealed that 16c may possess a mechanism of action through DNA binding that is similar to that of CCNU (lomustine). DNA binding studies indicated that 14g and 16c interact with the calf thymus DNA by intercalation and groove binding, respectively. Compounds 14g, 16c and 16a displayed strong binding affinities to DNA, EGFR and VEGFR-2 receptors. None of the active compounds showed cytotoxicity against human red blood cells.

Pathogenesis and in vivo interactions of human Streptococcus agalactiae isolates in the Galleria mellonella invertebrate model.

Group B Streptococcus (GBS) is a gram positive bacterium colonizing the gastrointestinal and urogenital tracts in humans. However under certain conditions GBS invades leading to severe infections in neonates, pregnant women, immunocompromised patients and the elderly people. The precise mechanisms involved in the transition from colonizer to pathogen remain to be elucidated, however it has been suggested that environmental determinants may regulate gene expression resulting in GBS invasion. We have assessed the potential of the moth Galleria mellonella as a model to study the in vivo virulence and GBS interactions of invasive and noninvasive human isolates from our population. Temperature, pH and bacterial competition effects were examined in the model as well as the response of Galleria hemocytes to GBS infection. GBS strains were able to effectively grow and infect G. mellonella in a dose dependent manner with a (half-lethal dose) LD50 1 × 107 CFU after 24 h. GBS infection induced larva melanization with hemocyte vacuolation and depletion. Larval killing increased with environmental conditions such as temperature (37 °C) and pH (≥5.5-7.2). Bacterial interference assays showed a remarkable antagonistic effect of Lactobacillus gasseri (cells and filtrates) on GBS infection and significantly improved Galleria survival. The protective effect of L. gasseri was observed even at ratios similar to those of GBS colonization suggesting that L. gasseri modulation by its metabolic products is relevant. Exposure to L. gasseri acidic filtrates induced growth inhibition and prevented larva killing after infection with the hypervirulent GBS clone (a multiresistant clinical ST 17 strain). We showed that mechanisms mediating these effects are mainly pH dependent, however other mechanisms may have a role depending on inocula. We also found that G. mellonella infected with invasive human GBS isolates showed differential killing curves with higher killing rates after 24 h when compared to those considered colonizers or noninvasive isolates. Overall it has been shown that G. mellonella may be a representative in vivo model for baseline GBS studies. Given the potential effects over the hypervirulent strain, our findings support the use of L. gasseri in the GBS control strategies based on Lactobacillus formulations.

Effects of biocide exposure on P. Aeruginosa, E. coli and A. Baumannii complex isolates from hospital and household environments / Efectos de la exposición a biocidas en aislados ambientales de P. Aeruginosa, E. coli y del complejo A. Baumannii hospitalarios y de la comunidad

Background: Bacterial responses to biocide exposure and its effects on survival and persistence remain to be studied in greater detail. Aim: To analyse the viability and survival of environmental isolates from household and hospital settings after biocide exposure. Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chlorhexidine (CHxG), benzalkonium chloride (BAC) and triclosan (TC) were determined in isolates of Pseudomonas aeruginosa, Acinetobacter baumannii complex and Escherichia coli collected from hospital and house- holds environments. Viability was monitored after exposure and removal of biocides using agar cultures and flow cytometry. Findings: P. aeruginosa isolates showed greater tolerance for all biocides tested whereas A. baumannii complex and E. coli were less tolerant. When compared with reference strains, biocide tolerance was up to 8 to 13-fold higher for TC and BAC respectively. Flow cytometry showed that biocide exposure may induce viable but non-growing states in P. aeruginosa and E. coli isolates before becoming fully replicative. Changes in the susceptibility profile in one isolate of A. baumannii complex were observed after biocide exposure. Discussion: Bacteria isolates from hospital and households were able to recover after biocide exposure at bactericidal concentrations favouring persistence and spread of biocide-tolerant strains. This study reinforces that cleaning compliance should be monitored by non-culture based tests. Novel formulations in cleaning and disinfection protocols should be revisited in hospitals harbouring P. aeruginosa and A. baumannii multidrug resistant isolates.

Introducción: El efecto de la exposición a biocidas en las poblaciones bacterianas, su viabilidad y persistencia requieren de estudios detallados. Objetivo: analizar la viabilidad y persistencia de bacterias de ambientes hospitalarios y domésticos posterior a la exposición a biocidas. Materiales y Métodos: En un estudio experimental in vitro se determinó la concentración inhibitoria mínima (CIM) y la concentración bactericida (CBM) para chlorhexidina (CHxG), cloruro de benzalconio (BAC) y triclcosan (TC) en aislados de Pseudomonas aeruginosa (10), el complejo Acinetobacter baumannii (5) y Escherichia coli (5) obtenidos de ambientes hospitalarios y domésticos. La viabilidad y susceptibilidad bacteriana después de la exposición y remoción del biocida fue evaluada por citometria de flujo y cultivo. Resultados: Independiente de su procedencia P. aeruginosa presentó mayor tolerancia a todos los biocidas. El complejo A. baumannii y E. coli fueron hasta 8 a 13 veces más tolerantes a BAC y TC que las cepas de referencia. Se observó que la exposición a biocidas altamente efectivos induce formas viables no replicativas en P. aeruginosa y E. coli. Un aislado del complejo A baumannii presentó cambios en el perfil de susceptibilidad posterior a la exposición. Discusión: Aislados tanto de ambiente hospitalario como de la comunidad pueden recuperarse después de la exposición a concentraciones bactericidas de los biocidas favoreciendo la persistencia y diseminación de bacterias no replicativas. Por lo anterior métodos alternativos al cultivo deben utilizarse en el seguimiento de protocolos de limpieza y desinfección. Los tiempos de recuperación de la viabilidad bacteriana deben tenerse en cuenta en la formulación de protocolos para erradicar y/o controlar cepas hospitalarias de P. aeruginosa o A. baumannii multirresistentes.

Emerging trends in invasive and noninvasive isolates of Streptococcus agalactiae in a Latin American hospital: a 17-year study.

BACKGROUND: Streptococcus agalactiae or group B Streptococcus (GBS) has been recognized as a lethal pathogen in neonates worldwide. S. agalactiae infections also severely affect pregnant women and immunosuppressed adults with substantial attributable morbidity and mortality. However, in Latin America, studies on the epidemiology and behaviour of S. agalactiae infections remain limited. METHODS: To better understand the behaviour of S. agalactiae infections in our region, we conducted a retrospective study to phenotypically describe S. agalactiae isolates collected in one of the largest hospitals in Colombia at two time periods: 1994-2001 and 2004-2012. The isolates were identified by biochemical analysis and tested for antimicrobial susceptibility. RESULTS: In 1994-2001 a total of 201 S. agalactiae isolates were found in urine 38.3%, vaginal exudates 27.8%, soft tissue 12.9%, and blood 8.5%. Susceptibility to ampicillin or penicillin was 94% whereas resistance to erythromycin and clindamycin were 2.8% and 5.2% respectively. In total 46 culture-positive cases of invasive infections were reported, 11 (24%) in neonates and 35 (76%) in adults. In 2004-2012 a total of 671 isolates were found in urine 47.8%, vaginal exudates 32.6%, soft tissue 2.7% and blood 9%. Susceptibility rates to ampicillin and penicillin were 98% whereas resistance to erythromycin and clindamycin were 12.5% and 9.4%. A total of 95 severe infections were reported: 12 (12.6%) were in neonates, 5 (5.3%) in children and 78 (82.1%) in adults. Over the 17-year study period the averaged prevalence of invasive S. agalactiae isolates was 17.4%. The estimated incidence for neonatal infections was 1.34 per 1000 livebirths (0.99 × 1000 livebirths for early- onset disease and 0.35 × 1000 livebirths for late- onset disease) whereas for non-pregnant adults the estimated incidence was 0.75 × 1000 admissions. CONCLUSIONS: A remarkable increase in bloodstream infections in immunosuppressed adults and a shift to early neonatal S. agalactiae infections were seen over time. We also found an increase in S. agalactiae resistance to erythromycin and clindamycin during the study period, and the emergence of penicillin-nonsusceptible isolates. Our findings are consistent with the global trends described elsewhere, reinforcing the need for S. agalactiae control measures in our region.