Impact of Antibiotic Use on Carbapenem Resistance in Pseudomonas aeruginosa: Is There a Role for Antibiotic Diversity?

In this study, we aimed to evaluate the relationship between the rates of resistance of Pseudomonas aeruginosa to carbapenems and the levels and diversity of antibiotic consumption. Data were retrospectively collected from 20 acute care hospitals across 3 regions of Switzerland between 2006 and 2010. The main outcome of the present study was the rate of resistance to carbapenems among P. aeruginosa. Putative predictors included the total antibiotic consumption and carbapenem consumption in defined daily doses per 100 bed days, the proportion of very broad-spectrum antibiotics used, and the Peterson index. The present study confirmed a correlation between carbapenem use and carbapenem resistance rates at the hospital and regional levels. The impact of diversifying the range of antibiotics used against P. aeruginosa resistance was suggested by (i) a positive correlation in multivariate analysis between the above-mentioned resistance and the proportion of consumed antibiotics having a very broad spectrum of activity (coefficient = 1.77; 95% confidence interval, 0.58 to 2.96; P < 0.01) and (ii) a negative correlation between the resistance and diversity of antibiotic use as measured by the Peterson homogeneity index (coefficient = -0.52; P < 0.05). We conclude that promoting heterogeneity plus parsimony in the use of antibiotics appears to be a valuable strategy for minimizing the spread of carbapenem resistance in P. aeruginosa in hospitals.

Poor value of pulsed-field gel electrophoresis to investigate long-term scale epidemiology of methicillin-resistant Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) is widely used for epidemic investigations of methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we evaluated its use in a long-term epidemiological setting (years to few decades, country to continent level). The clustering obtained from PFGE patterns after SmaI digestion of the DNA of 20 strains was compared to that obtained using a phylogenetic typing method (multiprimer RAPD). The results showed that the analysis of small PFGE bands (10-85kb) correlates better with multiprimer RAPD than the analysis of large PFGE bands (>85-700kb), suggesting that the analysis of small bands would be more suitable for the investigation of long-term epidemiological setting. However, given the technical difficulties to obtain a good resolution of these bands and the putative presence of plasmids among them, PFGE does not appear to be a method of choice for the long-term epidemiology analysis of MRSA.

PPARβ/δ prevents endoplasmic reticulum stress-associated inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism.

AIM/HYPOTHESIS: Endoplasmic reticulum (ER) stress, which is involved in the link between inflammation and insulin resistance, contributes to the development of type 2 diabetes mellitus. In this study, we assessed whether peroxisome proliferator-activated receptor (PPAR)β/δ prevented ER stress-associated inflammation and insulin resistance in skeletal muscle cells. METHODS: Studies were conducted in mouse C2C12 myotubes, in the human myogenic cell line LHCN-M2 and in skeletal muscle from wild-type and PPARβ/δ-deficient mice and mice exposed to a high-fat diet. RESULTS: The PPARβ/δ agonist GW501516 prevented lipid-induced ER stress in mouse and human myotubes and in skeletal muscle of mice fed a high-fat diet. PPARβ/δ activation also prevented thapsigargin- and tunicamycin-induced ER stress in human and murine skeletal muscle cells. In agreement with this, PPARβ/δ activation prevented ER stress-associated inflammation and insulin resistance, and glucose-intolerant PPARβ/δ-deficient mice showed increased phosphorylated levels of inositol-requiring 1 transmembrane kinase/endonuclease-1α in skeletal muscle. Our findings demonstrate that PPARβ/δ activation prevents ER stress through the activation of AMP-activated protein kinase (AMPK), and the subsequent inhibition of extracellular-signal-regulated kinase (ERK)1/2 due to the inhibitory crosstalk between AMPK and ERK1/2, since overexpression of a dominant negative AMPK construct (K45R) reversed the effects attained by PPARβ/δ activation. CONCLUSIONS/INTERPRETATION: Overall, these findings indicate that PPARβ/δ prevents ER stress, inflammation and insulin resistance in skeletal muscle cells by activating AMPK.

The ubiquitin-proteasome system in prostate cancer and its transition to castration resistance.

Prostate cancer is the most common carcinoma in the male population. In its initial stage, the disease is androgen-dependent and responds therapeutically to androgen deprivation treatment but it usually progresses after a few years to an androgen-independent phase that is refractory to hormonal manipulations. The proteasome is a multi-unit protease system that regulates the abundance and function of a significant number of cell proteins, and its inhibition results in cancer cell growth inhibition and apoptosis and is already exploited in the clinic with the use of proteasome inhibitor bortezomib in multiple myeloma. In order to be recognized by the proteasome, a target protein needs to be linked to a chain of the small protein ubiquitin. In this paper, we review the role of ubiquitin-proteasome system (UPS) in androgen receptor-dependent transcription as well as in the castration resistant stage of the disease, and we discuss therapeutic opportunities that UPS inhibition offers in prostate cancer.

Bacterial overgrowth during treatment with omeprazole compared with cimetidine: a prospective randomised double blind study.

BACKGROUND: Gastric and duodenal bacterial overgrowth frequently occurs in conditions where diminished acid secretion is present. Omeprazole inhibits acid secretion more effectively than cimetidine and might therefore more frequently cause bacterial overgrowth. AIM: This controlled prospective study compared the incidence of gastric and duodenal bacterial overgrowth in patients treated with omeprazole or cimetidine. METHODS: 47 outpatients with peptic disease were randomly assigned to a four week treatment regimen with omeprazole 20 mg or cimetidine 800 mg daily. Gastric and duodenal juice were obtained during upper gastrointestinal endoscopy and plated for anaerobic and aerobic organisms. RESULTS: Bacterial overgrowth (> or = 10(5) cfu/ml) was present in 53% of the patients receiving omeprazole and in 17% receiving cimetidine (p < 0.05). The mean (SEM) number of gastric and duodenal bacterial counts was 6.0 (0.2) and 5.0 (0.2) respectively in the omeprazole group and 4.0 (0.2) and 4.0 (0.1) in the cimetidine group (p < 0.001 and < 0.01; respectively). Faecal type bacteria were found in 30% of the patients with bacterial overgrowth. Basal gastric pH was higher in patients treated with omeprazole compared with cimetidine (4.2 (0.5) versus 2.0 (0.2); p < 0.001) and in patients with bacterial overgrowth compared with those without bacterial overgrowth (5.1 (0.6) versus 2.0 (0.1); p < 0.0001). The nitrate, nitrite, and nitrosamine values in gastric juice did not increase after treatment with either cimetidine or omeprazole. Serum concentrations of vitamin B12, beta carotene, and albumin were similar before and after treatment with both drugs. CONCLUSIONS: These results show that the incidence of gastric and duodenal bacterial overgrowth is considerably higher in patients treated with omeprazole compared with cimetidine. This can be explained by more pronounced inhibition of gastric acid secretion. No patient developed signs of malabsorption or an increase of N-nitroso compounds. The clinical significance of these findings needs to be assessed in studies with long-term treatment with omeprazole, in particular in patients belonging to high risk groups such as HIV infected and intensive care units patients.

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.

Comparative Histopathology of Biomphalaria glabrata, B. tenagophila and B. straminea with Variable Degrees of Resistance to Schistosoma mansoni Miracidia

A comparative histopathological study of three snails species - Biomphalaria glabrata, B. tenagophila and B. straminea - which had been infected with Schistosoma mansoni miracidia revealed similar qualitative features, consisting of areas of sporocyst proliferation and differentiation associated with reactive host reaction, at the time they were actively eliminating great number of cercariae. However, in specimens that were exposed to miracidia but failed to eliminate cercariae later on, different histopathological pictures were observed in different snail species. While B. glabrata exhibited frequent focal (granulomatous) proliferation of amebocytes in several organs, B. tenagophila and B. straminea only rarely showed such reactive changes, suggesting that the mechanism of resistance to miracidial infection probably follows different pathways in the snail species studied

Sensitivity of Leishmania spp. to Glibenclamide and 4-Aminopiridine: A Tool for the Study of Drug Resistance Development

We have demonstrated that Leishmania spp. grown as promastigotes, are sensitive to the K+ channel inhibitors 4-aminopyridine and glibenclamide. Their host cells, the macrophages, are not affected by similar concentrations of the drugs. We have also initiated the molecular characterization of the mechanisms involved in the development of drug resistance to glibenclamide by the parasite. Therefore, we have selected experimentally and begun to characterize the Venezuelan Leishmania (Leishmania) strain, NR resistant to glibenclamide [NR(Gr)]. The analysis of genomic DNA evidenced the existence of a fragment which apparently is amplified in NR(Gr). The fragment recognized by the pgpA probe, related to the Leishmania P-glycoprotein family and which was originally isolated from L. tarentolae, showed a size polymorfism between the sensitive and the resistant strain. These results suggest that the development of resistance to glibenclamide in the strain NR(Gr) might be associated with the amplification of the ltpgpA or related gene(s)

Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

HIV type 1 drug resistance among naive patients from Venezuela.

In this study, we characterize proviral DNA of 20 HIV-1 asymptomatic antiretroviral-naive patients from Venezuela in env, gag, and pol genes regions. Results from both env/gag HMA subtyping and phylogenetic analysis of pol partial sequences led to the description of clade B in all cases. Nevertheless, the high prevalence of polymorphisms was particularly evident among the protease sequences. A 10% prevalence of major resistance mutations to RTIs was found. Our data also suggested that the protease polymorphisms I62T and V77T could be considered as molecular markers of the subtype B local epidemic. In addition, we show how proviral DNA can be used as a reliable tool to follow trends of resistance mutation transmission.

A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.