Efflux Pumps

Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10 8 cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 g of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene-50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.

The accumulation of cisplatin is decreased in many cisplatin-resistant cell lines, and an active efflux pump for cisplatin exists in some of them, but it has not yet been identified. In this study, we transfected the copper-transporting P-type ATPase cDNA (ATP7B) into human epidermoid carcinoma KB-3-1 cells. The transfectant, KB/WD cell line, which overexpressed the P-type ATPase, ATP7B, was resistant to both cisplatin (8.9-fold) and copper (2.0-fold). The accumulation of cisplatin in KB/WD cells was lower than in mock-transfected KB/CV cells, and the efflux of cisplatin from KB/WD cells was enhanced compared with KB/CV cells. KB/WD cells were sensitive to other heavy metals, such as antimony, arsenate, arsenite, cadmium, and cobalt. ATP7B was overexpressed in cisplatin-resistant prostate carcinoma PC-5 cells but not in the parental PC-3 cells and the revertant PC-5R cells. ATP7B may be involved in cisplatin resistance in some tumors.

Treatment of multidrug-resistant tuberculosis has become one of the major problems in public health. Understanding the molecular mechanisms of drug resistance has been central to tuberculosis research in recent times. DNA microarray technology provides the platform to study the genomic variations related to these mechanisms on a comprehensive level. To investigate the role of efflux pumps in drug resistance, we have constructed a custom DNA microarray containing 25 drug efflux pump genes of Mycobacterium tuberculosis (Indian Patent file no. 2071=DEL=2007) and monitored changes in the expression of these genes on exposure of common antituberculous drugs. Expression profiling of efflux pump genes in multidrug-resistant M. tuberculosis isolates showed overexpression of 10 genes following exposure to various anti-tuberculous drugs. Although two of these genes (Rv3065 and Rv2938) have already been reported to be active drug efflux pumps in M. tuberculosis in earlier studies, the increased activities of other eight efflux pump genes (Rv1819, Rv2209, Rv2459, Rv2477c, Rv2688, Rv2846, Rv2994, and Rv3728) have been demonstrated in multidrug-resistant isolates by us for the first time. After confirmation of differential expressions of these genes by real-time reverse transcription polymerase chain reaction, it was observed that a simultaneous overexpression of efflux pump genes Rv2459, Rv3728, and Rv3065 was associated with resistance to the combination of isoniazid and ethambutol, and these drugs, along with streptomycin, were identified to group together, where efflux-mediated drug resistance appears to be important in M. tuberculosis and follows a constant pattern of induction in multidrug-resistant isolates. Isoniazid and ethambutol combination was also found to be affected in 10% (6=60) of the clinical isolates in the presence of carbonyl cyanide m-chloro phenylhydrazone in resazurin microtitre plate assay, supporting the role of efflux pumps in the resistance to these drugs. Overexpression of two of the genes (Rv2477 and Rv2209) has also been observed with ofloxacin stress in M. tuberculosis.

Chronic otitis externa in dogs is often associated with Pseudomonas aeruginosa infection. Fluoroquinolones are often used for treating such infections. Fluoroquinolone resistance mechanisms were characterized in 10 strains of P. aeruginosa isolated from chronic canine otitis externa. Nine out of ten strains harbored a mutation in the gyrA gene and presented an overexpression of efflux pump(s). There was a good correlation between the lipophilicity of the fluoroquinolone being tested and the effect of the efflux pump inhibitor in the final MIC. Therefore, both mechanisms, mutation in the gyrA gene and increased efflux pump(s), seem to play an important role in the acquisition of fluoroquinolone resistance in veterinary clinical isolates of P. aeruginosa. Levels of resistance to fluoroquinolones suggest that they could not be a good choice for systemic therapy of Pseudomonas otitis.

As part of a project to characterise plant-derived natural products that modulate bacterial multidrug resistance (MDR), bioassay-guided fractionation of a chloroform extract of the aerial parts of Rosmarinus officinalis led to the characterisation of the known abietane diterpenes carnosic acid (1), carnosol (2) and 12-methoxy-trans-carnosic acid. Additionally, a new diterpene, the cis A/B ring junction isomer of 12-methoxy-trans-carnosic acid, 12-methoxy-cis-carnosic acid , was isolated.

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

We have previously obtained, by exposure to near continuous increas ing concentrations of cisplatin, a panel of human ovarian cancer cell lines that exhibit a wide range of primary resistance to the drug (9- to >400- fold). These cells had strikingly increased (4- to 50-fold) levels of gluta- thione (GSH) as compared with the drug-sensitive cells of origin (A. K.

To investigate phenotypically and genotypically the presence of MDR efflux pumps in 21 clinical isolates of Staphylococcus haemolyticus collected over a period of 10 years. MICs of different antibiotics and biocides were determined by the broth dilution method in the presence/absence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an efflux pump inhibitor. PCR followed by sequencing was performed to detect the qac genes that encode for antiseptic resistance. Clonal relationships were determined by PFGE SmaI patterns using a standard protocol. All the isolates were resistant to gentamicin, 15 to erythromycin, 18 to ciprofloxacin, 7 to chloramphenicol and 1 to tetracycline. They showed higher susceptibility to antibiotics when they were exposed to CCCP. The MICs of ethidium bromide, SDS and benzalkonium chloride were also decreased, whereas the MIC of triclosan was decreased in only four isolates in the presence CCCP. Of the 21 isolates, qacA/B was detected in 5 isolates, smr in a...

From the dichloromethane extract of aerial parts of Ferula vesceritensis (Apiaceae), 11 sesquiterpene derivatives were isolated. Among them five were compounds designated as 10-hydroxylancerodiol-6anisate, 2,10-diacetyl-8-hydroxyferutriol-6-anisate, 10-hydroxylancerodiol-6-benzoate, vesceritenone and epoxy-vesceritenol. The six known compounds were identified as feselol, farnesiferol A, lapidol, 2-acetyl-jaeschkeanadiol-6-anisate, lasidiol-10-anisate and 10-oxo-jaesckeanadiol-6-anisate. All the structures were determined by extensive spectroscopic studies including 1D and 2D NMR experiments and mass spectroscopy analysis. Two of the compounds, the sesquiterpene coumarins farnesiferol A and feselol, bound to the model recombinant nucleotide-binding site of an MDR-like efflux pump from the enteropathogenic protozoan Cryptosporidium parvum.

A series of new 5,5-diphenylhydantoin derivatives with various amineealkyl terminal fragments at N1position were synthesized. Then a series of twenty-eight compounds with the same hydantoin scaffold were evaluated for their potency to combat bacterial MultiDrug Resistance (MDR). Intrinsic antibacterial activities were first evaluated. As these compounds showed no direct activity on bacteria, their influence on minimal inhibitory concentration (MIC) of nalidixic acid was tested in two strains of Enterobacter aerogenes: the reference-strain ATCC-13048 and the CM-64 strain which over-produces AcrAB-TolC efflux pump. The compounds showed moderate-or low-anti-MDR properties. According to SAR-studies, hit compounds containing 2-methoxyphenylpiperazine at N1-terminal fragment and methylcarboxyl acid one at N3-position of hydantoin have been identified for further microbiological studies and pharmacomodulations to develop efflux pump inhibitors.

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

The purpose of this exploratory study is to better understand the current dynamics of the Malaysian market for smartphone and the usage behaviors of consumers. This paper presents the result of a survey on the trend of smartphone from the perspective of end consumers. The data was collected from 1814 respondents across major cities in Malaysia. This study has looked into the familiarity of users towards smartphones, choices of smartphone brand and service providers, and most importantly the determinants that influence their purchasing decision. Additionally, the consumers' preferences on smartphone specifications such as design, computing power, operating platform, and price were investigated. Furthermore, consumers' usage behaviors such as using smartphone for email, web browsing, gaming, and document reading were examined. The statistics presented provides fundamental information regarding the trends in the smartphone market and usage behaviors in Malaysia. Such information are useful for academics for the development of future works in the field, whereas for smartphone manufacturers, application developers and other stakeholders, they are able to plan their direction in the Malaysian smartphone market.

The ISO 9001 (2000) define "Quality" as the ability of a set of inherent properties and characteristics of a product, system or process to fulfill requirements of customers and other interested parties. The "Plan de Calidad de Obra (PCO)" is an instrument that follows to get the necessary quality in Civil Works contracts. Graphical documentation generated during construction project elaboration or even during Civil Engineering works execution with planes, makes obligatory a meticulous treatment of this information that results transcendental in the consecution of the definitive products (projects or works) quality objective. In this work it's shown the principal graphical documentation procedures defined in the PCO attending to the edition, modification, distribution, storage, and other relevant aspects related to graphical information.

Anticancer drug resistance almost invariably emerges and poses major obstacles towards curative therapy of various human malignancies. In the current review we will distinguish between mechanisms of chemoresistance that are predominantly mediated by ATP-driven multidrug resistance (MDR) efflux transporters, typically of the ATP-binding cassette (ABC) superfamily, and those that are independent of such drug efflux pumps. In recent years, multiple nanoparticle (NP)-based therapeutic systems have been developed that were rationally designed to overcome drug resistance by neutralizing, evading or exploiting various drug efflux pumps and other resistance mechanisms. NPs are being exploited for selective drug delivery to tumor cells, to cancer stem/tumor initiating cells and/or to the supportive cancer cell microenvironment, i.e. stroma or tumor vasculature. Some of these NPs are currently undergoing preclinical in vivo studies as well as advanced stages of clinical evaluation with promising results. Nanovehicles harboring a payload of therapeutic drug combinations for the selective targeting and elimination of tumor cells as well as the simultaneous overcoming of mechanisms of drug resistance are a subject of intense research efforts, some of which are expected to enter clinical trials in the near future. In the present review we highlight novel approaches to selectively target cancer cells and overcome drug resistance phenomena, through the use of various nanometric drug delivery systems. In the near future, it is anticipated that innovative theragnostic nanovehicles will be developed which will harbor four major components: (1) a selective targeting moiety, (2) a diagnostic imaging aid for the localization of the malignant tumor and its micro-or macrometastases, (3) a cytotoxic, small molecule drug(s) or novel therapeutic biological(s), and (4) a chemosensitizing agent aimed at neutralizing a resistance mechanism, or exploiting a molecular "Achilles hill" of drug resistant cells. We propose to name these envisioned four element-containing nanovehicle platform, "quadrugnostic" nanomedicine. This targeted strategy holds promise in paving the way for the introduction of highly effective nanoscopic vehicles for cancer therapeutics while overcoming drug resistance.

Attenuation of Pseudomonas aeruginosa virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorumsensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the P. aeruginosa quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorumsensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections.

Ralph Brothers' parents both died of acute myocardial infarctions at an early age. Ralph inherited dyslipidemia and glucose intolerance, and his blood pressure is above normal. Determined to prevent an early death, he has altered his diet, initiated regular exercise, purchased glucose and blood pressure monitoring devices, and he also takes blood pressure medications regularly. He has a happy family and work life with a comfortable income.

Microorganisms capable of actively solubilizing and precipitating gold appear to play a larger role in the biogeochemical cycling of gold than previously believed. Recent research suggests that bacteria and archaea are involved in every step of the biogeochemical cycle of gold, from the formation of primary mineralization in hydrothermal and deep subsurface systems to its solubilization, dispersion and re-concentration as secondary gold under surface conditions. Enzymatically catalysed precipitation of gold has been observed in thermophilic and hyperthermophilic bacteria and archaea (for example, Thermotoga maritime, Pyrobaculum islandicum), and their activity led to the formation of gold-and silver-bearing sinters in New Zealand's hot spring systems. Sulphatereducing bacteria (SRB), for example, Desulfovibrio sp., may be involved in the formation of goldbearing sulphide minerals in deep subsurface environments; over geological timescales this may contribute to the formation of economic deposits. Iron-and sulphur-oxidizing bacteria (for example, Acidothiobacillus ferrooxidans, A. thiooxidans) are known to breakdown gold-hosting sulphide minerals in zones of primary mineralization, and release associated gold in the process. These and other bacteria (for example, actinobacteria) produce thiosulphate, which is known to oxidize gold and form stable, transportable complexes. Other microbial processes, for example, excretion of amino acids and cyanide, may control gold solubilization in auriferous top-and rhizosphere soils. A number of bacteria and archaea are capable of actively catalysing the precipitation of toxic gold(I/ III) complexes. Reductive precipitation of these complexes may improve survival rates of bacterial populations that are capable of (1) detoxifying the immediate cell environment by detecting, excreting and reducing gold complexes, possibly using P-type ATPase efflux pumps as well as membrane vesicles (for example, Salmonella enterica, Cupriavidus (Ralstonia) metallidurans, Plectonema boryanum); (2) gaining metabolic energy by utilizing gold-complexing ligands (for example, thiosulphate by A. ferrooxidans) or (3) using gold as metal centre in enzymes (Micrococcus luteus). C. metallidurans containing biofilms were detected on gold grains from two Australian sites, indicating that gold bioaccumulation may lead to gold biomineralization by forming secondary 'bacterioform' gold. Formation of secondary octahedral gold crystals from gold(III) chloride solution, was promoted by a cyanobacterium (P. boryanum) via an amorphous gold(I) sulphide intermediate. 'Bacterioform' gold and secondary gold crystals are common in quartz pebble conglomerates (QPC), where they are often associated with bituminous organic matter possibly derived from cyanobacteria. This may suggest that cyanobacteria have played a role in the formation of the Witwatersrand QPC, the world's largest gold deposit.

Organic-solvent-tolerant bacteria are a relatively novel group of extremophilic microorganisms. They overcome the toxic and destructive effects of organic solvents due to the presence of various adaptive mechanisms. Extensive studies done on the toluene tolerance of certain Pseudomonas strains have led to an understanding of the mechanisms of organic solvent tolerance involving novel adaptations such as the toluene efflux pumps, cis-trans isomerisation of membrane fatty acids, rapid membrane repair mechanisms, etc. Organic-solvent-tolerant mutants of Escherichia coli have been constructed and genes enhancing such tolerance characterised. However, there is practically no information available on the tolerance mechanisms of the reported Gram-positive organic-solvent-tolerant bacterial strains like Bacillus, Rhodococcus and Arthrobacter. This review discusses the general aspects of organic-solvent-tolerant bacteria, their history, biodiversity, mechanisms of tolerance and proposes certain probable adaptations of Gram-positive bacteria in tolerance to organic solvents.  2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Interpretative reading of the non-fermenting Gram-negative bacilli antibiogram Among non-fermenting Gram-negative rods, the most clinically important species are Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia, which are frequently multiresistant. P. aeruginosa resistance to beta-lactams depends on the production of chromosomal and plasmid-mediated beta-lactamases, altered permeability (loss of OprD porin is related to carbapenem-resistance) and active efflux pumps, particularly MexAB-OprM. In aminoglycoside-resistant strains the main mechanism of resistance is the production of inactivating enzymes; the efflux pump MexXY-OprM is also involved. Quinolone-resistance in P. aeruginosa is related to changes in topoisomerases, altered permeability and efflux pumps. The mechanisms of resistance of A. baumannii have not been well characterized, which makes interpretative reading of the antibiogram in this organism difficult. Resistance to beta-lactams is associated with the production of beta-lactamases and altered penicillin-binding proteins. Resistance to aminoglycosides has been related to modifying enzymes and resistance to quinolones to altered targets. S. maltophilia is resistant to carbapenems and other beta-lactams because of the production of two beta-lactamases (L-1 and L-2). Aminoglycoside-modifying enzymes have also been described in this species. In contrast to what is observed in other organisms, S. maltophilia resistance to quinolones has been mainly related to active efflux, rather than to target alterations.

In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effec- tive treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and (3H)palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833)

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were ␤-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were ␤-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 g/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins.

Pathogenic yeasts from the genus Candida can cause serious infection in humans particularly, in immunocompromised patients and are now recognized as major agents of hospital acquired (nosocomial) infections. In the recent years, there has been a marked increase in the incidence of treatment failures in candidiasis patients receiving long-term antifungal therapy, which has posed a serious problem in its successful use in chemotherapy. Candida cells acquire drug resistance (MDR) during the course of the treatment. The mechanisms of resistance to azole antifungal agents have been elucidated in Candida species and can be mainly categorized as (i) changes in the cell wall or plasma membrane, which lead to impaired drug (azole) uptake; (ii) alterations in the affinity of the drug target Erg11p (lanosterol 14µ-demethylase) especially to azoles or in the cellular content of Erg11p due to target site mutation or overexpression of the ERG11 gene; and (iii) the efflux of drugs mediated by membrane transport proteins belonging to the ATP-binding cassette (ABC) transporters, namely CDR1 and CDR2 or to the major facilitator superfamily (MFS) transporter, CaMDR1. Many such manifestations are associated with the formation of Candida biofilms including those occurring on devices like indwelling intravascular catheters. Biofilm-associated Candida show uniform resistance to a wide spectrum of antifungal drugs. A combination of different resistance mechanisms is responsible for drug resistance in clinical isolates of Candida species.

Attenuation of Pseudomonas aeruginosa virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorumsensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the P. aeruginosa quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorumsensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections.

The work presented in this thesis explores the potential role of bacteria in the arsenic cycle an agricultural soil from Scarlino (Tuscany), and a soil from Torviscosa (Fiuli), pyrite cinders contaminated. Both soils contained high levels of total arsenic. The soil of Scarlino contained 250 mg kg -1 , mainly associated to iron oxides (70% of the total), while the labile forms were 10% of the total. Total As in the soil of Torviscosa was 446 mg kg -1 , mainly present in the residual phase (50% of the total), while the labile forms represented 15% of the total.

Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the tod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD and ttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

This paper is an attempt to develop software framework for public library based and Webenabled multilingual community information services by utilizing open standards and open source software. It uses GSDL as DL software, PERL, PHP and Java as programming environment, Unicode as character encoding standard, SeamlessUK Application Profile as metadata schema, RFC-2731 as metadata encoding standard, and an array of FLOSS based multilingual tools for designing Bengali script based user interface, processing and retrieval environment.

The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged