Appropriateness of colonoscopy requests according to EPAGE-II in the Spanish region of Catalonia.

BACKGROUND: In a context of increasing demand and pressure on the public health expenditure, appropriateness of colonoscopy indications is a topic of discussion. The objective of this study is to evaluate the appropriateness of colonoscopy requests performed in a primary care (PC) setting in Catalonia. METHODS: Cross-sectional descriptive study. Out-patients >14 years of age, referred by their reference physicians from PC or hospital care settings to the endoscopy units in their reference hospitals, to undergo a colonoscopy. Evaluation of the appropriateness of 1440 colonoscopy requests issued from January to July 2011, according to the EPAGE-II guidelines (European Panel on the Appropriateness of Gastrointestinal Endoscopy). RESULTS: The most frequent indications of diagnostic suspicion requests were: rectal bleeding (37.46 %), abdominal pain (26.54 %), and anaemia study (16.78 %). The most frequent indications of disease follow-up were adenomas (58.1 %), and CRC (31.16 %). Colonoscopy was appropriate in 73.68 % of the cases, uncertain in 16.57 %, and inappropriate in 9.74 %. In multivariate analysis, performed colonoscopies reached an OR of 9.9 (CI 95 % 1.16-84.08) for qualifying as appropriate for colorectal cancer (CRC) diagnosis, 1.49 (CI 95 % 1.1-2.02) when requested by a general practitioner, and 1.09 (CI 95 % 1.07-1.1) when performed on women. CONCLUSIONS: Appropriateness of colonoscopy requests in our setting shows a suitable situation in accordance with recognized standards. General practitioners contribute positively to this appropriateness level. It is necessary to provide physicians with simple and updated guidelines, which stress recommendations for avoiding colonoscopy requests in the most prevalent conditions in PC.

Dissemination of extended-spectrum beta-lactamase-producing bacteria: the food-borne outbreak lesson.

OBJECTIVES: Commensal and opportunistic bacteria producing extended-spectrum beta-lactamases (ESBL-PB) have undergone a broad and rapid spread within the general population; however, the routes of dissemination have not been totally elucidated. The aim of this study was to determine whether individuals involved in an outbreak of acute gastroenteritis, in addition to the enteropathogenic microorganism, share an ESBL-PB as indirect demonstration of its transmission from a common food source. METHODS: From 2003 to 2004 in Barcelona, Spain, stool samples from 905 people involved in 132 acute gastroenteritis outbreaks and 226 food handlers related to the outbreaks were investigated. RESULTS: In 31 outbreaks, 58 diners carrying one or more ESBL-PB were detected. In 10 outbreaks, two or more diners shared the same ESBL-PB, and in four of them, the strain was shared with the food handlers. CONCLUSIONS: This study provides circumstantial evidence that foods can be a transmission vector for ESBL-PB, probably from two reservoirs, food animals and food handlers.

Immigration of Bacillus thuringiensis to bean leaves from soil inoculum or distal plant parts.

AIMS: We addressed the process of immigration of Bacillus thuringiensis from soil to leaves and its capacity to grow on bean diffusate medium (BDM), a medium designed to simulate the nutrient composition of the phylloplane. METHODS AND RESULTS: Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (<10 CFU cm(-2) of leaf). In addition, the number of cells of B. thuringiensis recovered decreased with increasing distance from the soil or from the inoculated leaves. Moreover, B. thuringiensis colonies did not grow well on BDM. CONCLUSIONS: This indicates that B. thuringiensis disperses poorly from the soil or the seed to the leaves or between leaves of the same plant under controlled conditions. Bacillus thuringiensis apparently has greater nutrient requirements than other bacterial species that are prominent inhabitants of the phylloplane. SIGNIFICANCE AND IMPACT OF THE STUDY: Finding the mechanisms that favour bacteria colonization on leaves will in turn help to improve the efficacy of biocontrol agents against the target pests.

Tannins from barks of Pinus caribaea protect Escherichia coli cells against DNA damage induced by gamma-rays.

This work was aimed to evaluate genotoxicity and antigenotoxicity activity against gamma-rays of a tannin fraction obtained from barks of Pinus caribaea, as well as to elucidate the antigenotoxic mechanisms involved in radioprotection by using different approaches as pre-, co- and post-irradiation cell treatments with plant extract. The tannin fraction was not genotoxic to Escherichia coli cells in experiments using different exposure times. This extract was antigenotoxic against gamma-rays when the cells were pre- or co-treated with this extracts, but not during post-irradiation treatments, suggesting a possibly antigenotoxic action through free radical scavenging mechanisms. The results are discussed in relation to the chemopreventive and therapeutic potential of the studied plant species.

The selection of resistance to and the mutagenicity of different fluoroquinolones in Staphylococcus aureus and Streptococcus pneumoniae.

Two quinolone-susceptible Staphylococcus aureus and five quinolone-susceptible Streptococcus pneumoniae isolates were used to obtain in-vitro quinolone-resistant mutants in a multistep resistance selection process. The fluoroquinolones used were ciprofloxacin, moxifloxacin, levofloxacin, gemifloxacin, trovafloxacin and clinafloxacin. The mutagenicity of these quinolones was determined by the Salmonella and the Escherichia coli retromutation assays. All quinolone-resistant Staph. aureus mutants had at least one mutation in the grlA gene, while 86.6% of quinolone-resistant Strep. pneumoniae mutants had mutations in either or both the gyrA and parC genes. Moxifloxacin and levofloxacin selected resistant mutants later than the other quinolones, but this difference was more obvious in Staph. aureus. Accumulation of the fluoroquinolones by Staph. aureus did not explain these differences, since levofloxacin and moxifloxacin accumulated inside bacteria to the same extent as clinafloxacin and trovafloxacin. The results also showed that moxifloxacin and levofloxacin had less mutagenic potency in both mutagenicity assays, suggesting a possible relationship between the selection of resistance to quinolones and the mutagenic potency of the molecule. Furthermore, gemifloxacin selected efflux mutants more frequently than the other quinolones used. Thus, the risk of developing quinolone resistance may depend on the density of the microorganism at the infection site and the concentration of the fluoroquinolone, and also on the mutagenicity of the quinolone used, with moxifloxacin and levofloxacin being the least mutagenic.

Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines.

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S. typhimurium) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA>2-AA>2-AF>9-AP>1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.

Virulence of Pasteurella multocida recA mutants.

In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a recA mutant was constructed and used in studies of virulence and competition in relation to wild-type strain. To achieve this, firstly, the recA gene was isolated and sequenced, showing an Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest identity with the Haemophilus influenzae RecA protein. Further, the recA mutant was constructed, by inactivating this gene by single recombination of a suicide plasmid containing an internal region of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD(50), the time of death of infected animals, and a failure to compete with the wild-type strain in mixed infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in competition were more a consequence of the long lag phase rather than a direct effect of the inactivation of the recA gene on genes involved in virulence.

Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases.

To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.

Radioprotective effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothioronicadenosin-5-triphosphate (adeturon) in gamma-irradiated Escherichia coli cells.

The effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate (adeturon) in the induction of Escherichia coli SOS response promoted by gamma-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in gamma-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by gamma-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed.

Activation of arylamines to mutagenic product(s) by two in vitro plant systems.

Plant activation of three isomers of phenylenediamine o- m- and p-phenylenediamine, has been studied. Two in vitro plant systems have been used: Persea americana S117 with mixed-function oxidase (MFO) and peroxidase activities, and Zea mays S9 which contains only peroxidase activity. As genetic endpoint, the classical Salmonella tester strains. TA98 and TA100, their derivatives with high O-acetyltransferase levels (YG1024 and YG1029, respectively) and TA98/1.8-DNP6, deficient in this enzyme, have been assayed. Of the three isomers studied, only m-PDA was activated to mutagenic product(s) by both plant systems. This activation required the bacterial O-acetyltransferase activity to give frameshift mutagenic product(s), detected in TA98 and YG1024 strains. In all the assays the P americana system was more potent than the Z. mays system in activating m-PDA. A slight increase of the number of YG1029 revertants was detected when m-PDA was activated by P. americana, suggesting that this compound can be also converted into ultimate mutagenic product(s) that induce base-pair substitutions. m-PDA activation by Z. mays was dependent on the peroxidase activity of this system, but the activation produced by P. americana was totally dependent on MFOs, because a total inhibition of the mutagenic response was found when these activities were inhibited. In addition, the P. americana system was more potent in generating proximal mutagenic forms from m-PDA than S9 from non-induced rat liver, although S9 from Aroclor 1254-induced Sprague-Dawley male rats was the most potent system in the m-PDA activation. These results indicate that the P. americana system can be useful in determining the role of mixed-function oxidases in plant activation of xenobiotics.

Identification of a pKM101 region which confers a slow growth rate and interferes with susceptibility to quinolone in Escherichia coli AB1157.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.

Efficiency of MucAB and Escherichia coli UmuDC proteins in quinolone and UV mutagenesis in Salmonella typhimurium: effect of MucA and UmuD processing.

The role of MucAB and Escherichia coli UmuDC proteins in mutagenesis by 4-quinolone (4-Q) compared to that in UV mutagenesis has been studied in hisG428 Salmonella typhimurium strains. A low-copy plasmid carrying mucAB genes, but not umuDC, promotes reversion of the hisG428 mutation by the 4-Q ciprofloxacin. In contrast, a umuDC plasmid mediates the reversion of hisG428 by UV, although less efficiently than a mucAB one. In addition, a unique copy of mucAB genes is enough to promote UV mutagenesis, whereas, several copies of them are required to detect ciprofloxacin mutagenesis. Therefore, the mutagenic repair of quinolone damage by MucAB proteins is not a very efficient process. The presence of an umuD'C plasmid but not a mucA'B one, slightly increases the reversion of the hisG428 mutation by ciprofloxacin and this finding is further discussed. In contrast, MucA'B are still more active than UmuD'C proteins in UV mutagenesis. These results suggest that the enhanced processing of MucA compared to UmuD would not explain all functional differences between MucAB and UmuDC proteins in the error-prone DNA repair.

Analysis of the ciprofloxacin-induced mutations in Salmonella typhimurium.

The mutagenic events induced by ciprofloxacin, a potent antimicrobial agent, have been characterized. For this, a battery of His mutants of Salmonella typhimurium (hisG428, his G46, His C9070, and his G1775 targets) that detects the six possible transitions and transversions [Levin and Ames (1986): Environ Mutagen 8:9-28] and two additional His strains (hisC3076 and his D3052 targets) carrying frameshift mutations have been used. Our results indicate that GC-TA transversions are the major base-pair substitution induced by ciprofloxacin and that GC-At transitions are also produced, but to a lesser degree. However, we cannot discard the fact that At-Ta transversions are also induced. In addition, the data indicate that the mutational specificity of ciprofloxacin depends on the location of the target. Intragenic base-pair substitutions are the most frequent mutations at the hisG428 target when it is on the chromosome, whereas 3 or 6 base-pair deletions are the major mutagenic events when this target is on the plasmid pAQ1. We have shown that ciprofloxacin also induces deletions/insertions at the hisC3076 and hisD3052 frameshift targets. Therefore, this inhibitor of DNA gyrase promotes a wide pattern of mutations including different kinds of base-pair substitutions, 3 or 6 base-pair deletions, and insertions/deletions resulting in frameshifts. All of these mutagenic events require the MucAb proteins involved in the error-prone repair, with the exception of base-pair insertions/deletions at the hisD3052 target, which are independent of the presence of plasmid pKM101.

A plant metabolic activation system from Persea americana with cytochrome P450-dependent and peroxidase activities.

Microsomal fractions from different tissues of various plants (potato, cauliflower, aubergine, avocado pear, courgette, cucumber, banana, kiwi and strawberry) were prepared and their content of cytochrome P-450 (cyt-P450) determined. A S117 fraction from Persea americana (avocado pear) presented the highest content of cyt-P450. As a consequence of these data, we have developed and characterized this fraction as a plant metabolic activation system. The P. americana S117, used in this work, contains 0.75 +/- 0.04 mg of protein per ml, 0.788 +/- 0.078 nmol of cyt-P450 per mg of protein and has a peroxidase activity of 0.036 +/- 0.005 (nmol tetraguaiacol/micrograms protein/min). The P. americana cyt-P450 remained stable during at least 60 days, stored at -80 degrees C. This fraction activated 2-aminofluorene to a mutagenic product in S. typhimurium TA98, while it had no effect on the benzo[a]pyrene activation. The treatment of the P. americana S117 with CO, the addition of diethyldithiocarbamate (DETC) or the absence of a NADPH-generating system in the activation mix, produced a partial inhibition of the 2-aminofluorene activation. Both peroxidase activity and a cyt-P450-dependent activity are assumed to be involved in the activation of this chemical mediated by P. americana S117.