Multidrug-resistant Ones Research Articles

Ferrocenyl-Coupled N-Heterocyclic Carbene Complexes of Gold(I): A Successful Approach to Multinuclear Anticancer Drugs.

Four gold(I) carbene complexes featuring 4-ferrocenyl-substituted imidazol-2-ylidene ligands were investigated for antiproliferative and antivascular properties. They were active against a panel of seven cancer cell lines, including multidrug-resistant ones, with low micromolar or nanomolar IC50 (72 h) values, according to their lipophilicity and cellular uptake. The delocalized lipophilic cationic complexes 8 and 10 acted by increasing the reactive oxygen species in two ways: through a genuine ferrocene effect and by inhibiting the thioredoxin reductase. Both complexes gave rise to a reorganization of the F-actin cytoskeleton in endothelial and melanoma cells, associated with a G1 phase cell cycle arrest and a retarded cell migration. They proved antiangiogenic in tube formation assays with endothelial cells and vascular-disruptive on real blood vessels in the chorioallantoic membrane of chicken eggs. Biscarbene complex 10 was also tolerated well by mice where it led to a volume reduction of xenograft tumors by up to 80 %.

Attributable mortality of ICU-acquired bloodstream infections: Impact of the source, causative micro-organism, resistance profile and antimicrobial therapy

ICU-acquired bloodstream infection (ICUBSI) in Intensive Care unit (ICU) is still associated with a high mortality rate. The increase of antimicrobial drug resistance makes its treatment increasingly challenging. We analyzed 571 ICU-BSI occurring amongst 10,734 patients who were prospectively included in the Outcomerea Database and who stayed at least 4 days in ICU. The hazard ratio of death associated with ICU-BSI was estimated using a multivariate Cox model adjusted on case mix, patient severity and daily SOFA. ICU-BSI was associated with increased mortality (HR, 1.40; 95% CI, 1.16-1.69; p=0.0004). The relative increase in the risk of death was 130% (HR, 2.3; 95% CI, 1.8-3.0) when initial antimicrobial agents within a day of ICU-BSI onset were not adequate, versus only 20% (HR, 1.2; 95% CI, 0.9-1.5) when an adequate therapy was started within a day. The adjusted hazard ratio of death was significant overall, and even higher when the ICU-BSI source was pneumonia or unknown origin. When treated with appropriate antimicrobial agents, the death risk increase was similar for ICU-BSI due to multidrug resistant pathogens or susceptible ones. Interestingly, combination therapy with a fluoroquinolone was associated with more favorable outcome than monotherapy, whereas combination with aminoglycoside was associated with similar mortality than monotherapy. ICU-BSI was associated with a 40% increase in the risk of 30-day mortality, particularly if the early antimicrobial therapy was not adequate. Adequacy of antimicrobial therapy, but not pathogen resistance pattern, impacted attributable mortality.

Plasma medicine—current state of research and medical application

Plasma medicine means the direct application of cold atmospheric plasma (CAP) on or in the human body for therapeutic purposes. Further, the field interacts strongly with results gained for biological decontamination. Experimental research as well as first practical application is realized using two basic principles of CAP sources: dielectric barrier discharges (DBD) and atmospheric pressure plasma jets (APPJ). Originating from the fundamental insights that the biological effects of CAP are most probably caused by changes of the liquid environment of cells, and are dominated by reactive oxygen and nitrogen species (ROS, RNS), basic mechanisms of biological plasma activity are identified. It was demonstrated that there is no increased risk of cold plasma application and, above all, there are no indications for genotoxic effects. The most important biological effects of cold atmospheric pressure plasma were identified: (1) inactivation of a broad spectrum of microorganisms including multidrug resistant ones; (2) stimulation of cell proliferation and tissue regeneration with lower plasma treatment intensity (treatment time); (3) inactivation of cells by initialization of programmed cell death (apoptosis) with higher plasma treatment intensity (treatment time). In recent years, the main focus of clinical applications was in the field of wound healing and treatment of infective skin diseases. First CAP sources are CE-certified as medical devices now which is the main precondition to start the introduction of plasma medicine into clinical reality. Plasma application in dentistry and, above all, CAP use for cancer treatment are becoming more and more important research fields in plasma medicine. A further in-depth knowledge of control and adaptation of plasma parameters and plasma geometries is needed to obtain suitable and reliable plasma sources for the different therapeutic indications and to open up new fields of medical application.

Occurrence of OXA-48 Carbapenemase and Other β-Lactamase Genes in ESBL-Producing Multidrug Resistant Escherichia coli from Dogs and Cats in the United States, 2009-2013.

Objective: The aim of this study was to explore the occurrence and molecular characterization of extended-spectrum β-lactamases (ESBL), plasmid-mediated AmpC β-lactamase (pAmpC) and carbapenemases among ESBL-producing multidrug resistant (MDR) Escherichia coli from dogs and cats in the United States.Methods: Of 2443 E.coli isolated from dogs and cats collected between August 2009 and January 2013, 68 isolates were confirmed as ESBL-producing MDR ones. PCR and sequencing were performed to identify β-lactamases and plasmid-mediated quinolone resistance (PMQR) genes, and shed light on the virulence gene profiles, phylogenetic groups and ST types.Results: Phylogenic group D and B2 accounted for 69.1% of the isolates. 50 (73.5%) isolates carried CTX-M ESBL gene, and the most predominant specific CTX-M subtype identified was blaCTX−M−15 (n = 33), followed by blaCTX−M−1 (n = 32), blaCTX−M−123 (n = 27), blaCTX−M−9 (n = 19) and blaCTX−M−14 (n = 19), and blaCTX−M−123 was firstly reported in E. coli isolates in the United States alone or in association. Other β-lactamase genes blaTEM, blaSHV, blaOXA−48, and blaCMY−2 were detected in 41.2, 29.4, 19.1, and 17.6% of 68 ESBL-producing MDR isolates, respectively. The blaTEM and blaSHV genes were classfied as ESBLs with the exception of the blaTEM−1 gene. Additionally, 42.6% (29/68) of isolates co-expressed blaCTX−M−15 and PMQR gene aac(6′)-Ib-c. The overall occurrence of virulence genes ranged from 11.8 (ireA) to 88.2% (malX), and most of virulence genes were less frequent among CTX-M-producing isolates than non-CTX-M isolates with the exception of malX and iutA. The 68 isolates analyzed were assigned to 31 STs with six being novel. Three pandemic clonal lineages ST131 (n = 10), ST648 (n = 9), and ST405 (n = 9) accounted for more than 41% of the investigated isolates, and ST648 and ST405 of phylogenetic D were firstly reported in E. coli from dogs and cats in the United States.Conclusion: blaCTX−M−123 of ESBLs and carbapenemase blaOXA−48 were firstly reported in ESBL-producing MDR E.coli from dogs and cats in the United States, and ST131, ST648, and ST405 were the predominant clonal groups.

In Vitro Antileukemia Activity of ZSTK474 on K562 and Multidrug Resistant K562/A02 Cells

Chronic myelogenous leukemia (CML) is a malignant hematological disorder mainly caused by the Bcr-Abl tyrosine kinase. While Bcr-Abl inhibitors including Imatinib showed antitumor efficacy on many CML patients, resistance was frequently reported in recent years. Therefore, novel drugs for CML are still expected. ZSTK474 is a specific phosphatidylinositol 3-kinase (PI3K) inhibitor that we identified. In the present study, the efficacy of ZSTK474, alone or in combination with Imatinib, on K562 CML cells as well as on its multidrug resistance counterpart K562/A02 cells, was investigated. ZSTK474 inhibited the cell proliferation with an IC50 of 4.69 μM for K562 and 7.57 μM for K562/A02 cells, respectively. Treatment by ZSTK474 resulted in cell cycle arrest in G1 phase, which might be associated with upregulation of p27, and downregulation of cyclin D1. ZSTK474 also inhibited phosphorylation of Akt and GSK-3β, which might be involved in the effect on the above cell cycle-related proteins. Moreover, combination of ZSTK474 and Imatinib indicated synergistic effect on both cell lines. In conclusion, ZSTK474 exhibited antileukemia activity alone, and showed synergistic effect when combined with Imatinib, on CML K562 cells as well as the multidrug resistant ones, providing a potential therapeutic approach for CML patients.

Spread of multidrug-resistant Escherichia coli within domestic aggregates (humans, pets, and household environment)

Abstract Advances in veterinary medicine have been associated with recourse to antimicrobial therapies, which favors the emergence of resistant bacteria, potentially able to spread globally. The aim of the present study was to elucidate the manner in which shared environments between pets receiving antimicrobial treatments and their owners could contribute to the spread of antimicrobial drug-resistant Escherichia coli . Three domestic aggregates, including pets, owners, and household environment were studied. Each core pet had a history of previous antimicrobial therapies. Overall, 231 E. coli isolates were recovered from pets' feces, urine, oral secretion, skin, and fur; and owners' hands and stool swabs. Commonly touched household objects and surfaces (light switches, door knobs, TV remote control, mobile phones, banister, refrigerator door handle, kitchen floor, pet beds, leash, food, and water recipients) were also sampled. All strains were analyzed by antimicrobial susceptibility testing. Subsequently, some isolates were selected for Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis analyses, to evaluate their genetic relatedness. Antimicrobial susceptibility tests displayed 20 different phenotypic patterns with an important representation of multidrug-resistant ones (75.0%). The 3 core dogs presented multidrug-resistant E. coli clones disseminated over various body sites. In 2 of 3 domestic aggregates, A and B, clonal disseminations among animals, owners, and household surfaces were also observed. Results confirmed the dissemination of multidrug-resistant E. coli within and through the household environment, highlighting the relevance of pets in the community spread of antimicrobial resistance.

Incidence of Active Tuberculosis Among Human Immunodeficiency Virus (HIV)-Positive Patients and Evaluation of Their Responses to Usual Anti-Tuberculosis Medications in Shiraz, South West of Iran

Background: Human Immunodeficiency Virus (HIV) makes infected cases prone to opportunistic infections like Tuberculosis (TB) due to impaired immunity of the body, especially Multi Drug Resistant (MDR) ones which are a major concern. With HIV outbreak starting late in the 20th century, the international health community is observing a huge rise in the incidence of this complex disease. Herein, we estimated the incidence of TB among HIV-positive individuals and their responses to anti-tuberculosis medications in Shiraz, Southwest of Iran. Materials and Methods: 840 HIV-positive patients were included in this cross-sectional study. During the first examination CD4+ count and PPD test was obtained, patients were checked for other symptoms too. Patients, if diagnosed with TB, received proper medication. If therapy failed, second-line therapy was prescribed for them. Type of resistance was studied and recorded. Patients continued their routine anti-viral therapy during the study. Results: Of 840 participants, 29 were diagnosed with Active TB (3.4%), 76% of them were diagnosed with PCR and culture and other with acid fast. Males were the majority of TB positives (82.8%). Most patients suffering from TB had CD4+ count lower than 200 (55.1%); 17.2% of the cases were MDR-TB. Conclusion: Low CD4+ count makes the patient vulnerable to TB. It is necessary to maintain patient’s immunity in order to treat and prevent tuberculosis; so, anti-viral therapies still play important roles in preventing TB in HIV-positive patients.[GMJ.2015;4(2):115-20]

Imidazolium salts as antifungal agents: strong antibiofilm activity against multidrug-resistant Candida tropicalis isolates.

The in vitro activity of the imidazolium salt C16 MImCl against planktonic and biofilm cells of multidrug-resistant isolates of Candida tropicalis was evaluated, both in solution and applied on a commercial catheter surface. This was determined by inhibition and susceptibility assays of biofilm and planktonic cells. In both cases, C16 MImCl prevented in vitro biofilm formation of C.tropicalis strains, including multidrug-resistant ones. Outstanding performances were observed, even at extremely low concentrations. Furthermore, this is the first report of the antifungal lock property of C16 MImCl, using a tracheal catheter as the test specimen to mimic a clinical in vivo condition. As such, C16 MImCl has been identified as a promising antimicotic pharmaceutical candidate for the treatment of candidiasis infections. The imidazolium salt 1-n-hexadecyl-3-methylimidazolium chloride (C16 MImCl) strongly prevents, in concentrations as low as 0·028μgml(-1) , the biofilm formation of multidrug-resistant Candida tropicalis isolates, either in solution or applied on the surface of commercial catheters. This presents an effective antimicotic candidate and alternative for invasive clinical procedure toolset asepsis.

El laboratorio de Microbiología en la vigilancia y el control de las infecciones nosocomiales

The most relevant activities of clinical microbiologist and the laboratory in the surveillance and the control of nosocomial infections (NI) are mainly focused on the collection, analysis and management of the information obtained in the Microbiology Laboratory; the design, development and validation of microbiological techniques, particularly rapid tests for the early detection of nosocomial pathogens, especially those multi-drug resistant ones, and the study of the genetic relationship between them. It also assists in the design of specific programs for the prevention of the NI, and the evaluation of their impact, as well as taking part in educational and training programs on topics related to NI. The management of laboratory resources, and communications with hospital information systems is also important.The most suitable tools for the control of NI include the correct identification at the species level of relevant nosocomial pathogens, analysis of the evolution of resistance to antimicrobials, monitoring sentinel organisms, active surveillance of carriers, and molecular epidemiology studies (genotyping). Prospectively typing of these pathogens, which has been achieved through advances in technology, and dissemination of molecular techniques, have a direct impact on the design of prevention and control interventions. To achieve the maximum performance with all these tools, it is essential to have a good communication strategy and an effective alert system.

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

New Methods to Clean ICU Rooms

Hospital-acquired infections (HAI) represent the most common adverse event in the intensive care unit (ICU). Their prevalence is high and they are associated with increased morbidity and mortality. The environment plays a central role in the transmission of hospital-acquired pathogens (HAP) and in the pathogenesis of HAI. Many bacteria, especially multidrug resistant ones, can survive for several months in the hospital environment in particular in areas close to the patients. It has been proven that pathogens are transmitted from the environment to the patients. Many studies have concluded that current cleaning methods are microbiologically ineffective. This failure concerns daily cleaning as well as terminal cleaning after patient discharge. It has been demonstrated that improvements in environmental cleaning are associated with a decrease in the rate of HAP and of HAI. New cleaning methods could enhance hospital cleaning efficiency. Three new technologies seem promising because they are microbiologically effective, easy and safe to use: (1) hydrogen peroxide vapor and (2) UV light decontamination are used for terminal cleaning. These techniques are effective even in difficultly accessible areas. (3) ultramicrofibers which can be associated with a copper-based biocide can be used for daily cleaning. Other methods such as ozone, steam or high-efficiency particulate air filtration are not efficient enough to be considered serious contenders for the improvement of the quality of the hospital environment. These new technologies have not been yet linked to a decrease in the prevalence and the incidence in HAP and HAI. It remains difficult to justify the extra-cost associated with these new methods until more studies can confirm their effectiveness in the management of HAI.

Cytotoxic Triterpenoid Constituents from Two Plants of the Family Meliaceae

For their interesting structures and significant biological activities, triterpenoids from plants of the family Meliaceae have been the research focus for several years. In our initial screening of the Meliaceae species, the extracts of Aglaia abbreviata C. Y. Wu and Aphanamixis grandifolia Bl exhibited some inhibitory activity against a panel of tumor cell lines. Phytochemical investigations on the two plants afforded six new dammarane-type triterpenoids, named aglaiabbreviatins A-F (1–6), along with twenty known triterpenoids from the stems of A. abbreviata, and twenty new tirucallane-type triterpenoids, named aphagranins A-T (7–26), along with ten known triterpenoids from the stem bark of A. grandifolia. Their structures were elucidated mainly by spectroscopic evidence. Of them, compound 9 has a maleimide ring in the side-chain; compound 19 incorporates a unique pyrrole ring, representing the first protolimonoid with such a moiety. All the new isolates were evaluated for their cytotoxicity against several tumor cell lines including multidrug resistant ones. Compounds 3–6, 11, 14–16 were found to have cytotoxicity to the tumor cell lines used, especially, compounds 6, 14, and16 showed the most potent activity.

Terpenyl-Purines from the Sea

Agelasines, asmarines and related compounds are natural products with a hybrid terpene-purine structure isolated from numerous genera of sponges (Agela sp., Raspailia sp.). Some agelasine analogs and related structures have displayed high general toxicity towards protozoa, and have exhibited broad-spectrum antimicrobial activity against a variety of species, including Mycobacterium tuberculosis, and also an important cytotoxic activity against several cancer cell lines, including multidrug-resistant ones. Of particular interest in this context are the asmarines (tetrahydro[1,4]diazepino[1,2,3-g,h]purines), which have shown potent antiproliferative activity against several types of human cancer cell lines. This review summarizes the sources of isolation, chemistry and bioactivity of marine alkylpurines and their bioactive derivatives.

Caractérisation phénotypique et génotypique ( randomly amplified polymorphic DNA analysis et multiple-locus variable-number tandem-repeat analysis) de 96 isolats cliniques de Pseudomonas aeruginosa à l’hôpital F. Bourguiba (Monastir, Tunisie)

Aim of the study Phenotypic and genotypic characterization of 96 clinical isolates of Pseudomonas aeruginosa recovered in a Tunisian teaching hospital during a 16-month period. Materials and methods All the isolates were characterized by serotyping, antimicrobial susceptibility typing and genotyping with randomly amplified polymorphic DNA (RAPD) analysis and multiple-locus variable-number tandem-repeat analysis (MLVA). Results Forty-one isolates out of 96 (43%) were recovered from two intensive care units (medical and chirurgical). Most of the isolates (48%) belonged to serotype O:11. Among the 13 antibiotypes, three multidrug resistant ones were mostly observed within the two intensive care units. Genotyping showed 83 RAPD types and 52 MLVA types. Isolates showing the same serotype could show different genotypes. A limited number of clusters was highlighted with MLVA typing, of which an outbreak of nine cases within the surgical intensive care unit. Conclusion Except this outbreak of nine cases, the heterogeneity observed for most of the P. aeruginosa isolates showed that outbreak situations were rare in the F. Bourguiba hospital during the study period. MLVA genotyping is a good tool for genotyping P. aeruginosa clinical isolates.

Risk factors for infection of the diabetic foot with multi-antibiotic resistant microorganisms

To investigate the risk factors for infection of the diabetic foot with multidrug resistant microorganisms. Amongst 102 diabetic patients with evidence of soft tissue infection of the foot who presented to our health center over a three year period, we investigated risk factors that might be predictive of multi-antibiotic resistance of the infecting organism. Of 102 patients with a diabetic foot wound, bacteria were cultured from 73, yielding a total of 104 isolates. The number of multidrug resistant isolates was 42 from 36 cases and the number of isolates other than multidrug resistant ones was 62 from 37 cases. Previous antibiotic therapy (p=0.002) and its duration (p=0.0001), frequency of hospitalization for the same wound (p=0.000), duration of hospital stay (p=0.000) and osteomyelitis (p=0.001) were significant risk factors for infections with multidrug resistant microorganisms. In conclusion, an appropriate antibiotic should be initiated promptly, wound perfusion should be effective, duration of hospital stay should be as short as possible and optimum hygiene should be provided during wound care to prevent infections of diabetic foot wound with multidrug resistant microorganisms.

Effects of miltefosine on membrane permeability and accumulation of [ 99mTc]-hexakis-2-methoxyisobutyl isonitrile, 2-[ 18F]fluoro-2-deoxy- d-glucose, daunorubucin and rhodamine123 in multidrug-resistant and sensitive cells

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [ 99mTc]-hexakis-2-methoxyisobutyl isonitrile ( 99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[ 18F]fluoro-2-deoxy- d-glucose ( 18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3–4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.

From molecular characteristics to cellularevents in apoptosis-resistant HL-60 cells

The ability to induce apoptosis in tumor cells is critical to elicit a positive response to cytotoxic chemo-therapy. In this study, we investigated the effect of the topoisomerase I inhibitors camptothecin and SN-38, known to cause an unusual form of DNA damage, on apoptotic pathways using the leukemic cell line HL-60 and its vincristine-resistant variant HL-60 VCR. Both camptothecin and SN-38 induced high levels of apoptosis in sensitive cells when compared to the multidrug-resistant ones. Interestingly, a higher BCL-2/BAX ratio was observed in HL-60 VCR at the basal state and during treatments. Moreover, these cells which did not exhibit Bcr-abl translocation or bcrp efflux pump, overexpressed topoisomerase I protein. The data provide evidence that BCL-2 protein could protect HL-60 VCR from mitochondrial membrane depolarization and block ROS production in these cells. Finally, our results suggest that dysregulation of proteins associated with DNA replication and apoptotic process could contribute to the multidrug-resistance phenotype.

Abnormal production of transforming growth factor β and interferon γ by peripheral blood cells of patients with multidrug-resistant pulmonary tuberculosis in Brazil

Objectives The aim of the present study was to determine the immune response profile that differentiates patients with newly diagnosed (non-treated) pulmonary tuberculosis from multidrug-resistant (MDR) ones, as well as from healthy, tuberculin positive individuals. Methods Lymphocytes proliferative response to non-specific mitogen (PHA) and PPD were evaluated by 3H thymidine incorporation and cytokines were quantified using an ELISA assay. Results Patients with active disease showed a diminished proliferative response to PHA and PPD, while multidrug-resistant patients showed a diminished proliferative response to PHA, but a normal response to PPD. The cytokine production of newly diagnosed patients was characterized by a diminished production of IFNγ and normal production of transforming growth factor (TGFβ), while MDR patients revealed a normal production of IFNγ accompanied by an increase in TGFβ. Conclusions The production of significant amounts of TGFβ in MDR patients leads to a poor immune response and may contribute to the resistance of tuberculosis patients to drugs.

Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study.

In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug-resistant. Genetic diversity was defined by PFGE. Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12) and anti-inflammatory cytokines (IL-10), of malondialdehyde and of procalcitonin. Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay. Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug-resistant isolates. To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte-mediated release of pro-inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF-alpha and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1beta and of IL-6 compared to stimulation by multidrug-resistant isolates. Similar differences were found for TNF-alpha and IL-8, but they were not statistically significant. Production of IL-10 and IL-12 was not detected after stimulation with any isolate. Levels of procalcitonin were similar after induction with either susceptible or multidrug-resistant isolates. Mean survival of animals was 7.56, 21.80 and 55.20 h, respectively, after challenge by the susceptible isolates and 28.89, 61.8 and more than 120 h, respectively, after challenge by the multidrug-resistant isolates. Differences of survival were accompanied by greater rodent monocyte-release of TNF-alpha and malondialdehyde after stimulation by the susceptible isolates compared to multidrug-resistant ones. It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa. These results correlate with in vivo evidence and might influence decision on therapeutics.

Antiproliferative and Apoptosis‐Inducing Effects of Hemin in Hepatoma Cells

Hemin is an extremely versatile molecule that may have cytotoxic or cytoprotective effects on certain cells. We investigated the effect of hemin on the growth of hepatoma cells, including the multidrug-resistant ones. Searching for new tools that interfere with the growth of hepatomas is an important area of clinical research. Cell viability and proliferation of drug-sensitive and multidrug-resistant hepatoma cell lines was determined using the trypan-blue exclusion test XTT/PMS and colony-forming ability assays. Apoptosis was assessed by confocal microscopy and DNA ladder assay. Hemin inhibited the proliferation and induced apoptosis in both drug-sensitive and multidrug-resistant hepatoma cells overexpressing functional P-glycoprotein. zVAD-fmk inhibited the hemin-induced decrease in cell viability, pointing to a role of caspases in hemin-induced apoptosis. The antiproliferative and apoptosis-inducing effects of hemin might be considered in the design of treatment for patients with hepatoma.