Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

Inhibition of thrombin,an unexplored function of retinoic acid

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC₅₀ values of 67μg/ml, 74μg/ml and 152μg/ml, respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.     

A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate

6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10³ – 10⁴ cells/0.2 ml growth medium/0.4 cm²) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 10⁵ colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 10⁵ colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.     

Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts

Antimicrobial and antiviral activities of the fractions from Scutia buxifolia stem bark and leaves were evaluated. Best antimicrobial results occurred with the ethyl acetate (EA) and n-butanolic (NB) fractions from the leaves against Micrococcus sp. (minimal inhibitory concentration—MIC = 62.5 μg/ml), and NB fraction from stem bark and leaves against Klebsiella pneumoniae and Enterococcus faecalis (MIC = 62.5 μg/ml). The most active fractions were selected and fractioned into silica column to perform an in vitro antibiofilm assay, which evidenced subfractions EA2 and EA3 as the more active against Candida albicans (biofilm inhibitory concentration—BIC = 582 ± 0.01 μg/ml) and Staphylococcus aureus (BIC = 360 ± 0.007 μg/ml), respectively. The NB (selectivity index—SI = 25.78) and the EA (SI = 15.97) fractions from the stem bark, and the EA (SI = 14.13) fraction from the leaves exhibited a potential antiviral activity towards Herpes Simplex Virus type 1 whereas EA2 and EA3 subfractions from leaves (SI = 12.59 and 10.06, respectively), and NB2 subfraction from stem bark (SI = 12.34) maintained this good activity. Phenolic acids and flavonoids (gallic acid, chlorogenic acid, caffeic acid, rutin, isoquercitrin, quercitrin and quercetin) were identified by HPLC and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that Scutia buxifolia has antibiofilm and anti-herpetic activities and that these properties are reported for the first time for this species.     

Antiplasmodial benzophenones from the trunk latex of Moronobea coccinea (Clusiaceae)

In an effort to find antimalarial drugs, a systematic in vitro evaluation on a chloroquine-resistant strain of Plasmodium falciparum (FcB1) was undertaken on sixty plant extracts collected in French Guiana. The methanol extract obtained from the latex of Moronobea coccinea exhibited a strong antiplasmodial activity (95% at 10 μg/ml). The phytochemical investigation of this extract led to the isolation of eleven polycyclic polyprenylated acylphloroglucinols (PPAPs), from which eight showed potent antiplasmodial activity with IC50 ranged from 3.3 μM to 37.2 μM.     

In vitro tetracycline susceptibility of Chlamydia trachomatis in clinical specimens

A novel approach to determine the tetracycline susceptibility of Chlamydia trachomatis directly from specimens without cell culture propagation and adaptation has been explored. Out of a total of 1290 genital specimens from a sexually transmitted disease clinic, 211 (16.4%) were positive for C. trachomatis. A tetracycline concentration of 0.032 g/ml completely inhibited the appearance of inclusions in all of the 211 positive specimens. Of the positive specimens, 120 (56.9%) and 18 (8.5%) respectively showed the presence of 1 to 9 and 100 or more inclusions per microtiter well in antibiotic free medium. Other antibiotics are being tested in the same manner.A part of this paper was presented at the Canadian Public Health Association, STD meeting in Ottawa, Canada, 28–29 October 1985     

Activation of neutrophils by Chlamydia trachomatis-infected epithelial cells is modulated by the chlamydial plasmid

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease world-wide. Chlamydia trachomatis primarily infects epithelial cells of the genital tract but the infection may be associated with ascending infection. Infection-associated inflammation can cause tissue damage resulting in female infertility and ectopic pregnancy. The precise mechanism of inflammatory tissue damage is unclear but earlier studies implicate the chlamydial cryptic plasmid as well as responding neutrophils. We here rebuilt the interaction of Chlamydia trachomatis-infected epithelial cells and neutrophils in-vitro. During infection of human (HeLa) or mouse (oviduct) epithelial cells with Chlamydia trachomatis, a soluble factor was produced that attracted neutrophils and prolonged neutrophil survival, independently of Toll-like receptor signaling but dependent on the chlamydial plasmid. A number of cytokines, but most strongly GM-CSF, were secreted at higher amounts from cells infected with plasmid-bearing, compared to plasmid-deficient, bacteria. Blocking GM-CSF removed the secreted pro-survival activity towards neutrophils. A second, neutrophil TNF-stimulatory activity was detected in supernatants, requiring MyD88 or TRIF independently of the plasmid. The results identify two pro-inflammatory activities generated during chlamydial infection of epithelial cells and suggest that the epithelial cell, partly through the chlamydial plasmid, can initiate a myeloid immune response and inflammation.     

Isolation and identification of antibacterial compounds from Thymus kotschyanus aerial parts and Dianthus caryophyllus flower buds

Aim of the studyThe aerial parts of Thymus kotschyanus Boiss. and Hohen. (Lamiaceae) and flower buds of Dianthus caryophyllus L. (Caryophyllaceae) have been traditionally implemented in the treatment of wounds, throat and gum infections and gastro-intestinal disorder by the indigenous people of northern Iraq, although the compounds responsible for the medicinal properties have not been identified. In this study, antibacterial compounds from both plants were isolated and characterized, and the biological activity of each compound was assessed individually and combined.Materials and methodsCompounds were isolated and characterized from the extracted essential oils of both plants using different spectral techniques: TLC, FTIR spectra and HPLC. The minimum inhibitory concentrations MIC values for the compounds were assessed individually and combined based on a microdilution and the checkerboard method in 96 multi-well microtiter plates.ResultsTwo known compounds were isolated from the essential oils of both plants and were identified as thymol and eugenol. The isolated compounds were investigated for their single and combined antibacterial activities against seven selected pathogenic bacteria; Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Thymol MIC values ranged from 15.6 to 250.0 μg/ml and B. cereus was found to be the most sensitive pathogen with a MIC value of 15.6 μg/ml. Eugenol achieved stronger MIC values against most tested pathogens and the best MIC value (15.6 μg/ml) was observed against B. cereus, L. monocytogenes and K. pneumoniae whereas, S. aureus, P. mirabilis and E. coli were inhibited with a MIC value of 31.2 μg/ml. Combination results had antibacterial enhancement against most pathogens and the best synergistic result was seen against P. mirabilis and E. coli.ConclusionsThe isolation of two antibacterial compounds from Thymus kotschyanus aerial parts and Dianthus caryophyllus flower buds validates the use of these species in the treatment of throat and gum infections, wound-healing and gastro-intestinal disorder.     

Evaluation of the Versant CT/GC DNA 1.0 Assay (kPCR) for the Detection of Extra-Genital Chlamydia trachomatis and Neisseria gonorrhoeae Infections

Screening for extra-genital Chlamydia trachomatis and Neisseria gonorrhoeae infections is a crucial component for sexually transmitted diseases management, even if at present days no commercial methods have been approved for use on pharyngeal and rectal specimens by the US FDA or have received the conformity CE marking. Here we report the analytical sensitivities of the Versant CT/GC 1.0 assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) on rectal and pharyngeal swabs, and an evaluation about the suitability for this assay with two widely used swab collection devices (E-Swab and eNAT, Copan, Brescia, Italy). The limits of detection for rectal and pharyngeal specimens with the Versant assay were 10 copies/ml and 1.0 copies/ml, for C. trachomatis and N. gonorrhoeae, respectively. False positive results due to the presence of non-gonococcal Neisseria species were excluded when clinical rectal and pharyngeal samples containing organisms identified as N. meningitidis, N. sicca, N. flavescens and N. subflava were tested. Due to its sensitivity and specificity, the Versant assay represents a good choice for the diagnosis of chlamydial and/or gonococcal infections not only in genito-urinary samples, but also on rectal and pharyngeal swabs.     

Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis

Purpose

Little is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.

Methods

Cultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.

Results

All eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin–8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).

Conclusions

This study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.     

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues ^1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur ^3,4. Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo ⁵. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans ^6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo ^8,9.For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.     

Lasalliapustulata lichen as possible natural antigenotoxic,antioxidant, antimicrobial and anticancer agent

The methanol extract of the lichen Lasallia pustulata was tested for genotoxic, antioxidant, antimicrobial and anticancer activities. We did this using a cytokinesis block micronucleus (MN) assay on peripheral blood lymphocytes, by measuring free radical and superoxide anion scavenging activity, reducing power, determining of total phenolic compounds and determining the total flavonoid content, measuring the minimal inhibitory concentration by the broth microdilution method against five species of bacteria and five species of fungi and by using the microculture tetrazolium test on FemX (human melanoma) and LS174 (human colon carcinoma) cell lines. As a result of this study, we found that the methanol extract of L. pustulata did not modify the frequency of the MN and nuclear division index in comparison to untreated cells (p > 0.05). These results revealed that the methanol extract had moderate free radical scavenging activity with IC₅₀ values of 395.56 μg/mL. Moreover, the extract tested had effective reducing power and superoxide anion radical scavenging. The values of the minimum inhibitory concentration against the tested microorganisms ranged from 0.625 to 20 mg/mL. In addition, the extract tested had strong anticancer activity against both cell lines with IC₅₀ values of 46.67 and 71.71 μg/mL.     

Synergy of aminoglycoside antibiotics by 3-Benzylchroman derivatives from the Chinese drug Caesalpinia sappan against clinical methicillin-resistant Staphylococcus aureus (MRSA)

The in vitro antimicrobial activities of three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) from Caesalpinia sappan L. (Leguminosae) were assayed, which mainly dealt with synergistic evaluation of aminoglycoside and other type of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by the three compounds through the Chequerboard and Time-kill curve methods. The results showed that Compounds 1–3 alone exhibited moderate to weak activity against methicillin-susceptible S. aureus (MSSA) and other standard strains by MICs/MBCs ranged from 32/64 to >1024/>1024 μg/ml, with the order of activity as 1 > 2 > 3. Chequerboard method showed significant anti-MRSA synergy of 1/Aminoglycosides (Gentamicin, Amikacin, Etimicin and Streptomycin) combinations with (FICIs)₅₀ at 0.375–0.5. The combined (MICs)₅₀ values (μg/ml) reduced from 32–128/16–64 to 4–8/4–16, respectively. The percent of reduction by MICs ranged from 50% to 87.5%, with a maximum of 93.8% (1/16 of the alone MIC). Combinations of 2 and 3 with Aminoglycosides and the other antibiotics showed less potency of synergy. The dynamic Time-killing experiment further demonstrated that the combinations of 1/aminoglycoside were synergistically bactericidal against MRSA. The anti-MRSA synergy results of the bacteriostatic (Chequerboard method) and bactericidal (time-kill method) efficiencies of 1/Aminoglycoside combinations was in good consistency, which made the resistance reversed by CLSI guidelines. We concluded that the 3-Benzylchroman derivative Brazilin (1) showed in vitro synergy of bactericidal activities against MRSA when combined with Aminoglycosides, which might be beneficial for combinatory therapy of MRSA infection.     

Inhibition of Candida albicans biofilm formation and yeast-hyphal transition by 4-hydroxycordoin

Candida albicans distinguishing features such as dimorphism and biofilm formation are thought to play a key role in oral tissue invasion and resistance to host defences and antifungal agents. In this study, we investigated the effect of 4-hydroxycordoin, a natural isopentenyloxychalcone, on growth, biofilm formation and yeast-hyphal transition of C. albicans. Serial dilutions of 4-hydroxycordoin in YNB medium were prepared in microplates to determine minimal inhibitory concentrations (MIC) and effects on biofilm formation for two strains of C. albicans. 4-Hydroxycordoin at up to 200 μg/ml had no effect on growth of C. albicans. Biofilm formation was strongly inhibited (>85%) by 4-hydroxycordoin at 20 μg/ml, while concentrations ranging from 50 to 200 μg/ml caused a significant inhibition of yeast-hyphal transition, as determined by microscopic observation. In conclusion, 4-hydroxycordoin exerts inhibitory effects on two important virulence factors of C. albicans: biofilm formation or yeast-hyphal transition. This suggests that 4-hydroxycordoin may have a therapeutic potential for C. albicans infections.     

Biological activity of Cymbopogon schoenanthus essential oil

IntroductionA number of plant species, including Cymbopogon schoenanthus, are traditionally used for the treatment of various diseases. C. schoenanthus is currently, traded in the Saudi markets, and thought to have medicinal value. This study aimed at investigating the biological activities of C. schoenanthus against both Gram-positive and Gram-negative bacteria and to identify its chemical ingredients.Materials and methodsThe inhibitory effects of water extracts of C. schoenanthus essential oils were evaluated against ten isolates of both Gram-positive and Gram-negative bacteria using the agar well diffusion and dilution methods. The minimum inhibitory concentration (MIC) was assayed using the Broth microdilution test on five of the ten isolates. The death rates were determined by the time kill assay, done according to the Clinical Laboratory Standards Institute (CLSI) guidelines. The chemical composition of the essential oils of the plant was performed using GC/MS.ResultsThe C. schoenanthus essential oil was effective against Escherichia coli, Staphylococcus aureus, methicillin-sensitive (MSSA) S. aureus (MRSA) and Klebsiella pneumoniae. The essential oil was not effective against Staphylococcus saprophyticus at the highest concentration applied of >150 μg/ml. The MIC values were as follows: 9.37 μg/ml for E. coli 4.69 μg/ml for S. aureus (MRSA), 2.34 mg/ml for MSSA and 2.34 μg/ml for K. pneumoniae. The time-kill assay indicated that there was a sharp time dependent decline in K. pneumoniae counts in the presence of the oil. This is in contrast to a gradual decline in the case of S. aureus under the same conditions. The eight major components of the essential oil were: piperitone (14.6%), cyclohexanemethanol (11.6%), β-elemene (11.6%), α-eudesmol (11.5%), elemol (10.8%), β-eudesmol (8.5%), 2-naphthalenemethanol (7.1%) and γ-eudesmol (4.2%).ConclusionThe results of the present study provide a scientific validation for the traditional use of C. schoenanthus as an antibacterial agent. Future work is needed to investigate and explore its application in the environmental and medical fields. In addition, to evaluating the efficacy of the individual ingredients separately to better understand the underlying mechanism.     

A new benzoic acid derivative from Piper diospyrifolium and its anti-Mycobacterium tuberculosis activity

The extraction by supercritical fluid (SFE-CO₂) from leaves of Piper diospyrifolium and chromatographic column purification afforded the isolation of a new benzoic acid derivative 4-methoxy-3-[(E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl-2-buten-1-yl)-benzoic acid (1). The chemical structure was elucidated on the basis of spectroscopic methods and comparison with literature data. SFE-CO₂ extracts and (1) were tested for their anti-Mycobacterium tuberculosis activities and cytotoxicities in J774G.8 macrophages. The compound (1) and SFE-CO₂ extracts exhibited moderate activities against M. tuberculosis H₃₇Rv with minimum inhibitory concentration (MIC) values of 125 μg/mL. The MIC values of M. tuberculosis clinical isolates ranged from 125 μg/mL to >250 μg/mL. The cytotoxicities results showed a selectivity index range from 0.6 to 1.0. Additional studies in structure activity-relationship as well as synergistic activity with antituberculous drugs should be conducted for a better evaluation of anti-mycobacterial activity of this compound.     

A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis

With an estimated 3–4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively while organisms known to co-inhabit the human urogenital tract react negatively.     

Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues

In an effort to develop potent anti-cancer chemopreventive agents that act on topoisomerase II, a novel series of bisindolylalkanes analogues such as 3,3′-(thiochroman-4,4-diyl)bis(1H-indole) are synthesized. Structures of all compounds are elucidated by ¹H NMR, ¹³C NMR and HRMS. Anti-proliferative activities for all of these compounds are investigated by the method of MTT assay on 7 human cancer lines. Most of them showed antitumor activities in vitro, the half maximal inhibitory concentration (IC₅₀) value is 7.798 μg/mL of 3a against MCF7. Compound 3a showed comparable topoisomerase II inhibitory activity to etoposide (VP-16) at 100 μM concentration. The rest of the compounds also showed varying degree topoisomerase II inhibitory activity.