Study of the Antibacterial Activity of Superhydrophilic and Superhydrophobic Copper Substrates against Multi-Drug-Resistant Hospital-Acquired Pseudomonas aeruginosa Isolates.

The global spread of multidrug-resistant (MDR) hospital-acquired pathogens is a serious problem for healthcare units. The challenge of the spreading of nosocomial infections, also known as hospital-acquired pathogens, including Pseudomonas aeruginosa, must be addressed not only by developing effective drugs, but also by improving preventive measures in hospitals, such as passive bactericidal coatings deposited onto the touch surfaces. In this paper, we studied the antibacterial activity of superhydrophilic and superhydrophobic copper surfaces against the P. aeruginosa strain PA103 and its four different polyresistant clinical isolates with MDR. To fabricate superhydrophilic and superhydrophobic coatings, we subjected the copper surfaces to laser processing with further chemosorption of fluorooxysilane to get a superhydrophobic substrate. The antibacterial activity of superhydrophilic and superhydrophobic copper surfaces was shown, with respect to both the collection strain PA103 and polyresistant clinical isolates of P. aeruginosa, and the evolution of the decontamination of a bacterial suspension is presented and discussed. The presented results indicate the promising potential of the exploitation of superhydrophilic coatings in the manufacture of contact surfaces for healthcare units, where the risk of infection spread and contamination by hospital-acquired pathogens is extremely high.

Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice.

The high prevalence of multidrug-resistant Acinetobacter baumannii has emerged as a serious problem in the treatment of nosocomial infections in the past three decades. Recently, we developed a new small-molecule inhibitor belonging to a class of 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT, previously called CL-55). FT effectively suppressed the T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella sp. without affecting bacterial growth in vitro. In this study, we describe that prophylactic use of FT for 4 days prior to challenge with resistant clinical isolates of A. baumannii (ABT-897-17 and 52TS19) suppressed septic infection in mice, resulting in improved survival, limited bacteraemia and decreased bacterial load in the organs of the mice. We show that FT had an inhibitory effect on A. baumannii biofilm formation in vitro and, to a greater extent, on biofilm maturation. In addition, FT inhibited Acinetobacter isolate-induced death of HeLa cells, which morphologically manifested as apoptosis. The mechanism of FT action on A. baumannii is currently being studied. FT may be a promising candidate for the development of a broad-spectrum anti-virulence drug to use in the prevention of nosocomial infections.

Chlamydia trachomatis Growth and Cytokine mRNA Response in a Prostate Cancer Cell Line.

In the present paper, we report that C. trachomatis can be efficiently propagated and affect mRNA expression for two major cytokines, relevant to tumor progression, in CWR-R1 cells, a malignant prostate cell line. CWR-R1 and McCoy cells, a classic cell line for chlamydial research, were grown and infected with C. trachomatis under similar conditions. Cell monolayers were harvested for RNA analysis and immunostaining with major outer membrane protein (MOMP) antibody at 24, 48, and 72 hours of the postinfection (hpi) period. It was shown that the infectious cycle of chlamydial pathogen in CWR-R1 cells resembles the progression of C. trachomatis infection in McCoy cells but with a few important differences. First of all, the initial stage of C. trachomatis propagation in CWR-R1 cells (24 hpi) was characterized by larger inclusion bodies and more intense, specific immunofluorescent staining of infected cells as compared with McCoy cells. Moreover, there was a corresponding increase in infective progeny formation in CWR-R1 cells along with mRNA for EUO, a crucial gene controlling the early phase of the chlamydial development cycle (24 hpi). These changes were more minimal and became statistically insignificant at a later time point in the infectious cycle (48 hpi). Altogether, these data suggest that the early phase of C. trachomatis infection in CWR-R1 cells is accompanied by more efficient propagation of the pathogen as compared with the growth of C. trachomatis in McCoy cells. Furthermore, propagation of C. trachomatis in CWR-R1 cells leads to enhanced transcription of interleukin-6 and fibroblast growth factor-2, genes encoding two important proinflammatory cytokines implicated in the molecular mechanisms of chemoresistance of prostate cancer and its ability to metastasize. The possible roles of reactive oxygen species and impaired mitochondrial oxidation in the prostate cancer cell line are discussed as factors promoting the early stages of C. trachomatis growth in CWR-R1 cells.

Lycopene presence in facial skin corneocytes and sebum and its association with circulating lycopene isomer profile: Effects of age and dietary supplementation.

Lycopene is a dietary antioxidant known to prevent skin photodamage. This study aimed to examine age-dependent presence of this carotenoid on the surface of the facial skin and in the serum as well as to measure the same parameters during supplementation with lycopene. Serum samples and samples from facial skin surface were obtained from 60 young (under 25 years old) and 60 middle-aged (over 50 years old) volunteers. Similar samples were taken from 15 middle-aged subjects during 4-week supplementation with lycopene (7 mg/day). Serum lycopene levels and isomer profiles were analyzed by HPLC. Lycopene in desquamated corneocytes and the sebum from facial skin surface was determined using lycopene-specific fluorescent monoclonal antibodies. The results demonstrated that there was no age-related difference in serum lycopene levels, but a higher proportion of (all-E)-lycopene was detected in the "young" group (37.5% vs 26.2% in the "middle-aged" group; p < 0.0001). "Young" volunteers also had a higher lycopene level in both corneocytes (p = 0.0071) and the sebum (p = 0.0139) from the skin surface. Supplementation with lycopene resulted in a sharp increase of lycopene concentrations in both serum and skin surface samples. There was also a clear change in the pattern of lycopene isomers in the serum manifested by a significant increase in the proportion of (all-E)-lycopene (from 22.1% to 44.0% after supplementation, p < 0.0001). It can be concluded that dietary supplementation with lycopene results in its accumulation in the serum and skin. This process is accompanied by significant changes in the circulating lycopene isomer profile which becomes similar to that typical for young individuals.

Non-Invasive Immunofluorescence Assessment of Lycopene Supplementation Status in Skin Smears.

Circulating lycopene level is negatively associated with the prevalence of cardiovascular disease, cancers (prostate and breast), type 2 diabetes mellitus, and aging. Traditionally, lycopene is measured in biological specimens by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry methods. Moreover, as we recently reported, tissue/cell lycopene depositions can be observed by the immunohistochemistry method with a newly developed monoclonal antibody (mAb) against lycopene. A main objective of this study is to evaluate the performance of a new noninvasive immunofluorescence (IF) lycopene quantification skin test with mAbs against lycopene versus HPLC lycopene assay of serum lycopene in volunteers subjected to lycopene supplementation which represents a novel approach to lycopene measurement methodology. For this purpose, 32 healthy volunteers, 30-40 years old, were supplemented with lycopene (n = 15) or placebo (n = 17) for a period of 4 weeks. It was found that lycopene supplementation leads to a significant increase in serum lycopene concentration after 2 and 4 weeks by 2.6- and 3.4-fold over control, respectively. This was accompanied by a concordant step-wise rise in IF staining of skin corneocytes and sebum, quantifiable by arbitrary IF scores. Placebo supplementation did not affect serum lycopene values or intensity of IF staining of the skin samples. There was 86.6% agreement in paired HPLC/IF variants for the intermediate time point and 80.0% agreement at the end of the study in the lycopene group. Intraclass correlation between paired values in this group was +0.49 for the 2-week time point and +0.63 for the end point. These results indicate that the new antibody-based skin assay can be used for rapid detection of lycopene deficiencies. Moreover, the noninvasive nature of the skin swab test would allow using it to monitor, optimize, and personalize lycopene supplementation protocol of risk groups in the general population.

Association with Monoclonal Antibody Promotes Intracellular Delivery of Lycopene.

Incubation of B10.MLM cells, a cell line of alveolar macrophages, with lycopene, a carotenoid, leads to an increase of lycopene content in their microsomal fraction. That increase was higher and developed faster when the cells were incubated with immune complexes formed by lycopene and mAb 6B9 (L-6B9 mAb), a monoclonal hapten-specific antibody raised against lycopene, as compared with dimethyl sulfoxide (DMSO)-dissolved lycopene (DMSO-L). Moreover, incubation of B10.MLM cells with L-6B9 mAb complexes was accompanied by more efficient accumulation of lipid droplets in the cultured cells and more significant inhibition of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase, a rate-limiting enzyme of cholesterol biosynthesis known to be targeted by lycopene. Additionally, there was a better inhibition of Chlamydia trachomatis infection in B10.MLM cells infected with the pathogen and incubated thereafter with L-6B9 mAb complexes as compared with DMSO-L. Altogether, the results suggest that association with monoclonal antibody promotes intracellular delivery of lycopene in cultured cells possibly through Fc-receptor mediated uptake.

Resveratrol Inhibits Propagation of Chlamydia trachomatis in McCoy Cells.

Resveratrol (RESV), an antifungal compound from grapes and other plants, has a distinct ability to inhibit the Chlamydia (C.) trachomatis developmental cycle in McCoy cells, a classic cell line used for chlamydial research. Inoculation of C. trachomatis with increasing amounts of RESV (from 12.5 to 100 µM) gave a dose-dependent reduction in the number of infected McCoy cells visualized by using monoclonal antibodies against chlamydial lipopolysaccharide. A similar trend has been observed with immunoassay for major outer membrane protein (MOMP). Furthermore, there was a step-wise reduction in the number of C. trachomatis infective progenies caused by the increasing concentrations of RESV. The ability of RESV to arrest C. trachomatis growth in McCoy cells was confirmed by a nucleic acid amplification protocol which revealed dose-dependent changes in mRNAs for different genes of chlamydial developmental cycle (euo, incA, and omcB). Although the precise nature of the antichlamydial activity of RESV is yet to be determined and evaluated in future studies, the observed effect of RESV on C. trachomatis infection was not related to its potential effect on attachment/entry of the pathogen into eukaryotic cells or RESV toxicity to McCoy cells. Similar inhibitory effect was shown for C. pneumoniae and C. muridarum.

Lycopene Inhibits Propagation of Chlamydia Infection.

Chlamydiaceae is a family of obligate intracellular pathogenic bacteria with similar developmental cycles and cell biology responsible for a wide range of diseases in different hosts including genital and eye inflammatory diseases, arthritis, and inflammatory diseases of the respiratory and cardiovascular systems. In the present paper, we report that lycopene, one of the main dietary carotenoids, which is present in tomato and some other fruits, has a strong inhibitory effect on C. trachomatis and C. pneumoniae infections in alveolar macrophages. This finding was documented by both immunofluorescence analysis and electron microscopy. It was noted that lycopene treatment inhibited intracellular phase of the chlamydial developmental cycle and resulted in a significant loss of infectious progeny. The antichlamydial effect of lycopene was also confirmed in a clinical setting. There was a significant reduction of IgG antibodies against C. pneumoniae in the serum of volunteers treated for a month with oral ingestion of 7 mg of lycopene. Additional studies are needed to further explore the antichlamydial activity of lycopene and its possible effect on C. pneumoniae in relation to antichlamydial activity of lycopene to mechanisms of atherosclerosis.

Generation and Application of Monoclonal Antibody Against Lycopene.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

A small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones inhibits intracellular growth and persistence of Chlamydia trachomatis.

Chlamydia trachomatis is one of the most common sexually transmitted pathogens in the world and often causes chronic inflammatory diseases that are insensitive to antibiotics. The type 3 secretion system (T3SS) of pathogenic bacteria is a promising target for therapeutic intervention aimed at bacterial virulence and can be an attractive alternative for the treatment of chronic infections. Recently, we have shown that a small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones produced through the chemical modification of the thiohydrazides of oxamic acids, designated CL-55, inhibited the intracellular growth of C. trachomatis in a T3SS-dependent manner. To assess the feasibility of CL-55 as a therapeutic agent, our aim was to determine which point(s) in the developmental cycle CL-55 affects. We found that CL-55 had no effect on the adhesion of elementary bodies (EBs) to host cells but significantly suppressed EB internalization. We further found that CL-55 inhibited the intracellular division of reticulate bodies (RBs). An ultrastructural analysis revealed loss of contact between the RBs and the inclusion membrane in the presence of CL-55. Finally, we found that our T3SS inhibitor prevented the persistence of Chlamydia in cell culture and its reversion to the infectious state. Our findings indicate that our T3SS inhibitor may be effective in the treatment of both productive and persistent infections.

Detection of C. trachomatis in the serum of the patients with urogenital chlamydiosis.

Extragenital chlamydial complications may be associated with systemic spread of infection, but haematogenous route for C. trachomatis dissemination has not been clearly demonstrated. Here we report that serum specimens obtained from patients with chlamydiosis contain elementary bodies of C. trachomatis shown by culture and immunogold electron microscopy. We have found that 31 of the 52 patients had serum precipitates which were infective to McCoy cells. Immunostaining revealed very small inclusions resembling those reported during persistent C. trachomatis infection in vitro. DNA specimens from 49 (out of 52) patients with chlamydiosis gave positive PCR readings. The viability of the pathogen present in the sera was confirmed by chlamydial RNA detection in the cell monolayer inoculated by the serum precipitates. By using DNA isolation protocol from 1 mL of serum and quantitative TaqMan PCR, it was estimated that bacterial load in patients' sera was 2 × 10(2)-10(3) GE/mL. These findings for the first time demonstrated that C. trachomatis can be disseminated directly by the plasma, independently from blood cell, which may represent a new possible pathway of the chronic infection development. Therefore, new methodological approaches for detection of C. trachomatis in the serum of patients with complicated and chronic chlamydiosis could be important in the diagnosis of the infection regardless of its anatomical localization.