Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking.

AIMS: To evaluate the ability of grapevine ecosystem fungi to degrade histamine, tyramine and putrescine in synthetic medium and in wines. METHODS AND RESULTS: Grapevine and vineyard soil fungi were isolated from four locations of Spain and were subsequently identified by PCR. A total of 44 fungi were evaluated for in vitro amine degradation in a microfermentation system. Amine degradation by fungi was assayed by reversed-phase (RP)-HPLC. All fungi were able to degrade at least two different primary amines. Species of Pencillium citrinum, Alternaria sp., Phoma sp., Ulocladium chartarum and Epicoccum nigrum were found to exhibit the highest capacity for amine degradation. In a second experiment, cell-free supernatants of P. citrinum CIAL-274,760 (CECT 20782) grown in yeast carbon base with histamine, tyramine or putrescine, were tested for their ability to degrade amines in three different wines (red, white and synthetic). The highest levels of biogenic amine degradation were obtained with histamine-induced enzymatic extract. CONCLUSION: The study highlighted the ability of grapevine ecosystem fungi to degrade biogenic amines and their potential application for biogenic amines removal in wine. SIGNIFICANCE AND IMPACT OF STUDY: The fungi extracts described in this study may be useful in winemaking to reduce the biogenic amines content of wines, thereby preventing the possible adverse effects on health in sensitive individuals and the trade and export of wine.

Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity.

AIMS: To evaluate the patterns of the production of antimicrobial compounds by diverse collection of actinomycetes isolated from different geographies under alternative conditions of pH and salinity in the media. METHODS AND RESULTS: Actinomycetes were grouped based on their method of isolation and their phenotype diversity was determined by total fatty acid analysis. A total of 335 representative isolates, including 235 Streptomyces species and 100 actinomycetes from other taxa, were screened for the production of antimicrobial activities against a panel of bacteria, filamentous fungi and yeasts, including some of clinical relevance. Production of antimicrobial activities was detected in 230 strains. In the case of the genus Streptomyces, 181 antimicrobial activities (77% of the tested isolates) were recorded. The activities observed among the other actinomycetes taxa were lower (49% of the tested isolates). CONCLUSIONS: The results of this study support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity present a significant capacity to produce compounds with antibacterial or antifungal activity. The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate that these actinomycetes strains isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity.

Microbial natural products as a source of antifungals.

The vast number and variety of chemotherapeutic agents isolated from microbial natural products and used to treat bacterial infections have greatly contributed to the improvement of human health during the past century. However, only a limited number of antifungal agents (polyenes and azoles, plus the recently introduced caspofungin acetate) are currently available for the treatment of life-threatening fungal infections. Furthermore, the prevalence of systemic fungal infections has increased significantly during the past decade. For this reason, the development of new antifungal agents, preferably with novel mechanisms of action, is an urgent medical need. A selection of antifungal agents in early stages of development, produced by micro-organisms, is summarized in this review. The compounds are classified according to their mechanisms of action, covering inhibitors of the synthesis of cell wall components (glucan, chitin and mannoproteins), of sphingolipid synthesis (serine palmitoyltransferase, ceramide synthase, inositol phosphoceramide synthase and fatty acid elongation) and of protein synthesis (sordarins). In addition, some considerations related to the chemotaxonomy of the producing organisms and some issues relevant to antifungal drug discovery are also discussed.

Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum.

AIMS: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed. METHODS AND RESULTS: An extract from a Trichoderma species showed a more potent activity in an agar-based assay against the null mutant fks1::HIS strain than against the wild-type strain, suggesting that it could contain a glucan synthesis inhibitor. The active component was identified as the known compound ergokonin A. The compound exhibited activity against Candida and Aspergillus species, but was inactive against Cryptococcus species. It induced alterations in the hyphal morphology of Aspergillus fumigatus. The identification of the producing isolate was confirmed by sequencing of the rDNA internal transcribed spacers and comparison with the sequences of other Trichoderma species. The analysis showed that the producing fungus had a high homology with other strains classified as Trichoderma longibrachiatum and its teleomorph Hypocrea schweinitzii. CONCLUSIONS: The antifungal activity spectrum of ergokonin A and the morphology alterations induced on A. fumigatus are consistent with glucan synthesis as the target for ergokonin A. The production of ergokonin A is not uncommon, but is probably restricted to Trichoderma species. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery that ergokonin A could be an inhibitor of glucan synthesis, having a structure very different to other inhibitors, increases the likelihood that orally active agents with this fungal-specific mode of action may be developed.

Diversity among clinical isolates of penicillin-resistant Streptococcus mitis: indication for a PBP1-dependent way to reach high levels of penicillin resistance.

A total of 12 non-epidemiologically related clinical isolates of Streptococcus mitis that showed different levels of resistance to penicillin were studied. Membrane-protein profiles and penicillin-binding protein (PBP) patterns showed a great polymorphism; and patterns of 4-7 PBPs, with sizes that ranged from approximately 101 kDa to approximately 40 kDa, were detected in each strain. No association could be found between PBP pattern and resistance level to penicillin among these isolates. Arbitrarily primed PCR confirmed the genetic diversity among this group of streptococci. One of the isolates of intermediate level of resistance to penicillin, which showed a PBP pattern similar to that of the high-resistance strains, was used as a laboratory model to analyse the mechanism underlying high-resistance acquisition by these strains. A 14-fold increase in penicillin resistance was obtained after a single selection step, which resulted in a decrease in penicillin affinity for PBP1. The size of this PBP (92 kDa) and the differences in PBP profiles of the penicillin-resistant clinical isolates suggest the existence in S. mitis of PBP-mediated mechanisms to acquire high-level resistance to penicillin, among which alterations in PBP1 seem to play a main role, in contrast to the PBP2X mediated mechanism described for other streptococci.

A randomized field trial of ACINDES: a child-centered training model for children with chronic illnesses (asthma and epilepsy).

UNLABELLED: A randomized field trial of a child-centered model of training for self-management of chronic illnesses was conducted of 355 Spanish-speaking school-aged children, between 6 and 15 years old, with moderate to severe asthma and epilepsy, in Buenos Aires, Argentina. The model, based on play techniques, consists of five weekly meetings of 8-10 families, with children's and parents' groups held simultaneously, coordinated by specially trained teachers and outside the hospital environment. Children are trained to assume a leading role in the management of their health; parents learn to be facilitators; and physicians provide guidance, acting as counselors. Group activities include games, drawings, stories, videos, and role-playing. Children and parents were interviewed at home before the program and 6 and 12 months after the program, and medical and school records were monitored for emergency and routine visits, hospitalizations, and school absenteeism. In asthma and epilepsy, children in the experiment showed significant improvements in knowledge, beliefs, attitudes, and behaviors compared to controls (probability of experimental gain over controls = .69 for epilepsy and .56 for asthma, with sigma2 = .007 and .016, respectively). Parent participants in the experiment had improved knowledge of asthma (39% before vs. 58% after) and epilepsy (22% before vs. 56% after), with a probability of gain = .62 (sigma2 = .0026) with respect to the control group. Similar positive outcomes were found in fears of child death (experimental 39% before vs. 4% after for asthma, 69% before vs. 30% after for epilepsy), as well as in disruption of family life and patient-physician relationship, while controls showed no change. Regarding clinical variables, for both asthma and epilepsy, children in the experimental group had significantly fewer crises than the controls after the groups (P = .036 and P = .026). Visits to physicians showed a significant decrease for those with asthma (P = .048), and emergency visits decreased for those with epilepsy (P = .046). An 18-item Children Health Locus of Control Scale (CHLCS) showed a significant increase in internality in experimental group children with asthma and epilepsy (P < .01), while controls did not change or performed worse 12 months after the program. School absenteeism was reduced significantly for those with asthma and epilepsy (for the group with asthma, fall/winter P = .006, and spring P = .029; for the group with epilepsy, P = .011). CONCLUSION: The program was successful in improving the health, activity, and quality of life of children with asthma and epilepsy. The data suggested that an autonomous (Piagetian) model of training is a key to this success, reinforcing children's autonomous decision making.

Screening of basidiomycetes for antimicrobial activities.

As a part of a screening programme developed to evaluate the antimicrobial activity of basidiomycetes, 317 isolates representing 204 species collected in Spain were screened against a range of human clinical pathogens and laboratory controls. Extracts from 45% of the isolates, representing 109 species, showed antimicrobial activity. Antibacterial activity was more pronounced than antifungal activity. The proportion of extracts from basidiomycetes showing antimicrobial activity was similar to or above that obtained for representative orders of Ascomycetes, such as Pezizales and Xylariales, but lower than that produced by members of the orders Diaporthales, Eurotiales, Hypocreales, Leotiales and Sordariales. Suprageneric taxa (orders and families) did not show pronounced differences in their antimicrobial activities though such differences were observed at the genus level, suggesting that the ability to produce these bioactive compounds is not homogenously distributed amongst the basidiomycetes. Isolates from some species showed large differences in their ability to produce metabolites with antimicrobial activity, possibly reflecting genetic differences at the infraspecific level.

Characterization of clinical isolates of beta-lactamase-negative, highly ampicillin-resistant Enterococcus faecalis.

We analyzed the penicillin-binding protein (PBP) profiles of two clinical isolates of Enterococcus faecalis for which ampicillin MICs were 32 and 64 micrograms/ml. Six PBPs were detected in both isolates, demonstrating an apparently increased amount of PBP 5 and decreased penicillin binding of PBPs 1 and 6. These results suggest that ampicillin resistance in the clinical isolates of E. faecalis described could be associated with alterations in different PBPs.

[The effect of family factors on school retardation in the Cartuja quarter of Granada]. / Influencia de algunos factores familiares en el retraso escolar en el barrio de Cartuja de Granada.

The possible influence of the family on school retardation was evaluated in the Cartuja quarter in Granada for the academic term 1988-89. The study was performed on 258 schoolchildren of the 7th and 8th primary courses in the four public schools from the quarter. Data were collected with a questionnaire including the following variables: school retardation, sex, number of siblings, ordinal number among siblings, educational level of the parents, and work of the mother outside the household. To evaluate family function the APGAR questionnaire was used. A remarkably high (45.7%) school retardation was found, higher than that in the rest of Spain. School retardation cannot be related with the family dysfunction detected by the familial APGAR. It was related, rather, with the child's sex, the educational level of the mother, the number of siblings and the ordinal place of the child among them.

Penicillin-binding protein 3 of Listeria monocytogenes as the primary lethal target for beta-lactams.

Penicillin-binding proteins (PBPs) of Listeria monocytogenes were detected by their ability to bind to [2,3-3H]benzylpenicillin. Five proteins with Mrs of 95,000, 84,000, 80,000, 76,000, and 49,000 were detected. PBPs 1 to 4 had a high affinity for [2,3-3H]benzylpenicillin and were relatively scarce (80 to 150 molecules per cell). In contrast, PBP 5 was more abundant (600 molecules per cell) but had a low affinity for [2,3-3H]benzylpenicillin. L. monocytogenes has a relatively high natural resistance to cephalosporins. Competition experiments showed that cephalosporins bound very poorly to PBP 3 but were good inhibitors of PBPs 1, 2, and 4, which were completely blocked at concentrations well below the MIC. Analysis of a spontaneous imipenem-resistant mutant revealed that resistance was likely due to an altered PBP 3 with a reduced affinity for [2,3-3H]benzylpenicillin. These results suggest that PBP 3 is a primary lethal target for beta-lactams in L. monocytogenes.

Penicillin binding proteins in Listeria monocytogenes.

Membranes of Listeria were obtained from protoplasts and treated with 125I-ampicillin as probe, at different concentrations. Eight bands, corresponding to proteins labelled with the probe were detected their molecular weight ranged from 38,000 to 100,000, being the predominant ones at 95,000; 85,000; 60,000; 49,000 and 38,000. The copy number of each PBP was also estimated. By means of competitive experiments the binding pattern of ampicillin, mecillinam, piperacillin, cefalotin, cefaloridine, cefoxitin, cefotaxime, azthreonam and imipenem was studied. The most effective binding was obtained with ampicillin, piperacillin and imipenem. Cefotaxime, and particularly cefoxitin, present an extremely low binding ability. The amount of antibiotic concentration preventing an effective label by the radioactive probe to the detected penicillin binding proteins seems to correlate with the lethal concentration of the different antibiotics on Listeria monocytogenes and explains the natural resistance of this genus to certain beta-lactamic compounds.

Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation.

DNA sequence analysis of the regions adjacent to the hlyA gene, which encodes listeriolysin O, an essential virulence factor of Listeria monocytogenes, revealed the presence of two open reading frames (ORFs): ORF D located 304 base pairs downstream from hlyA, and ORF U located 224 base pairs upstream from and in opposite direction to hlyA. Promoter mapping performed with RNAs extracted from cells growing exponentially in rich medium showed that the three ORFs are independently transcribed. hlyA is transcribed from two promoters separated by 10 base pairs (P1 hlyA and P2 hlyA). ORF U is transcribed in the opposite direction from an adjacent promoter. These two promoter regions are separated by a palindromic sequence T-T-A-A-C-A-A/T-T-G-T-T-A-A. This palindrome was also found upstream from the ORF D promoter, suggesting that all three genes are similarly regulated.

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.

Bleomycin-kanamycin resistance as a marker of the presence of transposon Tn5 in clinical strains of Escherichia coli.

The aminoglycoside modifying enzyme aminoglycoside 3'-phosphotransferase II (APH(3')II) is encoded for on transposon Tn5 by the aphA gene, in the same operon as the ble gene determining bleomycin resistance. To document this linkage 82 kanamycin-resistant Escherichia coli strains of clinical origin were studied; all 18 isolates presenting bleomycin-kanamycin resistance were shown by an enzymatic assay to produce APH(3')II, and the presence of Tn5 was demonstrated by gene hybridization. Similarly, bleomycin-kanamycin resistance was shown to be linked to APH(3')II production in Salmonella spp. The epidemiology of strains with Tn5-encoded APH(3')II may thus be studied, at least in Escherichia coli, by a simple diffusion test using bleomycin and kanamycin discs.

A genetic approach to demonstrate the role of listeriolysin O in the virulence of Listeria monocytogenes.

The locus of insertion of a transposon previously used to obtain a non-haemolytic avirulent mutant was identified: it is the structural gene encoding lisreriolysin O, the thiol-dependent haemolysin, now called hlyA. The gene was completely sequenced. The preliminary structural and functional study of the chromosomal region containing the gene indicates that hlyA belongs to a monocistronic transcriptional unit. If it is the case, the transposon insertion would have no major polar effect on downstream genes and would only affect hlyA expression. These results emphasize the importance of the haemolysin in the virulence of Listeria monocytogenes.

Towards a physical map of the Listeria chromosome: the pulsed field electrophoresis approach.

The possibility of the electrophoretic study of very large fragments of the Listeria chromosome (DNAs up to 1000 Kb) may help to the understanding of the physical organization and functional mapping of the entire genome of this organism. Several experiences were done to apply this technology to Listeria. Agarose inserts were prepared with intact cells and protoplasts, and lysis was induced in situ. Inserts with a convenient amount of DNA were cleaved in situ again by diffusing restriction enzymes into the agarose and submitted to one dimensional electric field that was periodically inverted, to cause changes of direction of the DNA fragments. We obtained a good band resolution of bands with Eco R1 and HindIII. Experiments are in progress to select other restriction enzymes leading to larger fragments. This technique must be combined with the use of blotting with known DNA sequences, such as the corresponding to the haemolysin, to arrive to a comprehensive map of the Listeria chromosome. Cloning of Listeria genes involved in several biochemical characteristics of Listeria, or at least a good collection of mutants will be quite necessary for the progress of such approach.

Reacquisition of virulence of haemolysin-negative Listeria monocytogenes mutants by complementation with a plasmid carrying the hlyA gene.

An haemolysin negative mutant of Listeria monocytogenes LO28 produced by insertion of Tn917 resulted in an avirulent derivative. The hlyA gene of the same strain was previously cloned in Escherichia coli and retransfered by transformation to the hly- derivative, after subcloning in the shuttle vector pMK4. The transformant strain (L828) reacquired an haemolytic activity at a similar level than the wild strain. The virulence of this hly+ transformant was estimated by determining the LD50 in Swiss mice infected intravenously. With increasing doses of bacteria a hly- control strain (transformant with only pMK4) appeared to be totally avirulent; however, no significant difference in virulence was found between the hly+ transformants and the wild strain. Seriol viable counts in the liver and spleen of infected mice demonstrated an increase in number of L828 hly+ transformants at 48 h, but the hly- control transformants were rapidly eliminated. These results confirm that the production of haemolysin is a major factor in the pathogenic capabilities of L. monocytogenes.