Inmaculada Herrero

Comparison of enterococcal populations in animals, humans, and the environment - a European study

The objectives of the present study were to generate knowledge of enterococcal populations in the food chain, by studying the population structure (in measures of abundance and diversity) among enterococci in different geographical regions and in different parts of the food chain, as well as the similarities between different enterococcal populations. Altogether, 2868 samples were collected from humans (healthy and hospitalised individuals and clinical isolates), animals (slaughterhouse carcasses and farm animals), and the environment (pig farms, sewage, and surface water) in four European countries-Sweden, Denmark, UK, and Spain. The samples were characterised with regard to presence and numbers of enterococci, and eight (for faecal samples) or 24 (for environmental samples) isolates per sample were phenotyped and preliminarily identified with the PhP-RF system. In total, more than 20,000 isolates were typed. A majority of the samples (77%) showed the presence of presumed enterococci. The diversities of enterococci in environmental samples were generally high, and also faecal samples normally showed presence of more than one enterococcal strain. The most common species found were Enterococcus faecium (33%), E. faecalis (29%), and E. hirae (24%), but different enterococcal populations differed in their species distribution. Clinical isolates, hospitalised patients, and hospital sewage in Sweden showed a clear dominance of E. faecalis (80%, 57%, and 54%, respectively) whereas healthy individuals and urban sewage contained less E. faecalis (39% and 40%, respectively). The species distribution among isolates from slaughterhouses varied between animal species and also between countries, but E. faecalis seemed to be mainly associated with broiler, and E. hirae with cattle and pigs. The results from the study have indicated a simplified method to study the diversity of bacterial populations. Instead of collecting many samples and analysing one or a few isolates per sample, it is possible to collect fewer samples and analyse several isolates per sample. Both approaches yielded similar information on the diversity of the populations. Another useful information was that since samples from hospital sewage, urban sewage, and manure contained enterococcal populations that reflected those in faecal samples of hospitalised patients, healthy humans, and animals, respectively, such samples may be used as pooled faecal samples and may replace cumbersome samplings from many individuals.

Presynaptic receptors and the control of glutamate exocytosis

When a typical glutamate-containing neurone fires, an action potential is propagated down the branching axon through more than a thousand varicosities. At each of these release sites the probability that a synaptic vesicle will be exocytosed into the synaptic cleft is individually controlled by means of presynaptic receptors: autoreceptors responding by positive or negative feedback to previously released transmitter, or heteroreceptors under the influence of other neurotransmitters or modulators. The simplest system in which to investigate presynaptic modulation is the isolated nerve terminal or synaptosome; studies with this preparation have revealed a complex interplay of signal-transduction pathways.

Rapid Desensitization of the Metabotropic Glutamate Receptor that Facilitates Glutamate Release in Rat Cerebrocortical Nerve Terminals

The metabotropic autoreceptor of glutamatergic nerve terminals from the cerebral cortex of adult rats has been characterized. Receptor activation involves a rapid and transient increase in diacylglycerol, which is sensitive to l‐2‐amino‐3‐phosphonopropionate (l‐AP3) and l‐2‐amino‐4‐phosphonobutanoic acid (l‐AP4) and is partially blocked by pertussis toxin. Protein kinase C (PKC) has a negative feedback control in this transduction pathway because the activation of the kinase, either by phorbol esters or by the endogenous diacylglycerol produced by the receptor, results in a reversible receptor desensitization, with loss of the ability to further facilitate glutamate release. It is concluded that the facilitatory metabotropic receptor located at the glutamatergic nerve endings belongs to the subclass coupled to phosphoinositide hydrolysis and that the rapid and use‐dependent desensitization of the facilitatory pathway may underlie a mechanism to prevent its permanent activation and thereby to avoid neurotoxicity.

Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions

ABSTRACT Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.

Antimicrobial resistance among enterococci from pigs in three European countries

ABSTRACT Enterococci from pigs in Denmark, Spain, and Sweden were examined for susceptibility to antimicrobial agents and copper and the presence of selected resistance genes. The greatest levels of resistance were found among isolates from Spain and Denmark compared to those from Sweden, which corresponds to the amounts of antimicrobial agents used in food animal production in those countries. Similar genes were found to encode resistance in the different countries, but the tet(L) and tet(S) genes were more frequently found among isolates from Spain. A recently identified transferable copper resistance gene was found in all copper-resistant isolates from the different countries.

Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors.

We have investigated the role of metabotropic glutamate receptors linked to phosphoinositide hydrolysis in the control of glutamate release in cerebrocortical nerve terminals. The activation of these receptors with the agonist 3,5-dihydroxyphenylglycine enhanced intrasynaptosomal diacylglycerol and facilitated both the depolarization-induced increase in the cytosolic free Ca2+ concentration and the release of glutamate. However, 5 min after receptor activation, a second stimulation of the pathway with the agonist failed to produce diacylglycerol and to facilitate glutamate release. Interestingly, during the period in which the diacylglycerol response was desensitized, a strong agonist-induced inhibition of Ca2+entry and glutamate release was observed. This change in the presynaptic effects of 3,5-dihydroxyphenylglycine is reversible since 30 min after the first stimulation, the agonist-induced inhibition of release disappeared, whereas both the production of diacylglycerol and the facilitation of glutamate release were recovered. The tonic elevation of the extracellular glutamate concentration from basal levels (0.8 μm) up to 5 μm also produced the switch from facilitation to inhibition in the receptor response. The existence of this activity-dependent switch in the presynaptic control of glutamate release suggests that release facilitation is limited to conditions under which an appropriate clearance of synaptic glutamate exists, probably to prevent the neurotoxic accumulation of glutamate in the synapse.

A Decrease in [Ca2+]c but not in cAMP Mediates L‐AP4 Inhibition of Glutamate Release: PKC‐mediated Suppression of this Inhibitory Pathway

We investigated the mechanism of the inhibition of glutamate release by L‐2‐amino‐4‐phosphonobutyrate (L‐AP4) in cerebrocortical nerve terminals from young rats (3 weeks of age). The Ca2+‐dependent release of glutamate was reduced by L‐AP4 in a concentration‐dependent manner. This inhibitory effect was prevented by pertussis toxin, insensitive to staurosporine and associated with a reduction both in the depolarization‐evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]c) and in forskolin‐stimulated cAMP formation. However, the reduction in [Ca2+]c but not in cAMP seemed to be responsible for the decrease in release, since inhibition by L‐AP4 can also be observed in the absence of detectable changes in CAMP. The inhibitory modulation by L‐AP4 was suppressed by the activation of protein kinase C with phorbol esters. The nerve terminals from young rats also exhibited a facilitatory pathway of glutamate release which was mediated by protein kinase C. Interestingly, stimulation of this pathway with the glutamate agonist (1 S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate in the presence of arachidonic acid also abolished the inhibitory action of L‐AP4. The dominance of the facilitatory pathway in its interaction with the L‐AP4‐mediated inhibitory control may provide some clues to understand the presynaptic changes during synaptic plasticity.

cAMP-dependent Facilitation of Glutamate Release by β-Adrenergic Receptors in Cerebrocortical Nerve Terminals

We have investigated the presence of a cAMP-protein kinase A-dependent pathway in cerebrocortical nerve terminals and its role in the modulation of glutamate release. The activation of adenylyl cyclase with forskolin enhances intrasynaptosomal cAMP and induces Ca2+-dependent glutamate release. The membrane permeant analogue dibutyryl cAMP mimics this facilitatory effect, whereas the inactive compound 1,9-dideoxyforskolin is without effect. This cAMP-induced facilitation is consistent with the induction of spontaneous action potentials that are abolished by the Na+ channel blocker tetrodotoxin and by reducing nerve terminal excitability with arachidonic acid. We have also demonstrated that a β-adrenergic receptor is linked to this pathway because isoproterenol increases cAMP levels and glutamate release, and both actions are antagonized by the receptor antagonist propanolol and the protein kinase A inhibitors H89 and 8-chloroadenosine 3′,5′-monophosphorothioate ((Rp)-isomer). The finding that the increase in cytoplasmic free Ca2+ concentration induced by synaptic activity reduces the concentration of agonist required to maximally activate adenylyl cyclase suggests that this enzyme may act as a coincidence detector, integrating glutamatergic neurotransmission and noradrenaline release.

Surveillance of antimicrobial resistance in Escherichia coli strains isolated from pigs at Spanish slaughterhouses

Antimicrobial resistance can make the efficient treatment of bacterial infections in humans and animals more difficult. Antimicrobial use in food animals may be one of the factors contributing to resistance. The Spanish surveillance network VAV has established a baseline of antimicrobial resistance in Escherichia coli strains from healthy pigs. Minimum inhibitory concentration and patterns of resistance to antimicrobials used in animals and humans were determined for 205 faecal strains isolated in a sampling frame of four slaughterhouses in Spain from 220 pigs in 1998. Higher levels of resistance were seen against antimicrobial agents authorised for use in food animals especially tetracycline, sulphonamides, trimethoprim and amoxycillin. All isolates were susceptible to antimicrobials employed mainly in humans such as ceftazidime, cefotaxime, imipenem, aztreonam and amikacin.

Developmental change from inhibition to facilitation in the presynaptic control of glutamate exocytosis by metabotropic glutamate receptors

We have addressed the role of presynaptic metabotropic glutamate receptors in the control of glutamate release from cerebrocortical nerve terminals. The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid enhances the release evoked by a submaximal depolarization in the presence of low concentrations of arachidonic acid and in a staurosporine-sensitive manner. In contrast, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid and L(+)-2-amino-4-phosphonobutyrate inhibit the release evoked by a maximal depolarization, in the absence of arachidonic acid and by a staurosporine-insensitive mechanism. Interestingly, the effects of the metabotropic glutamate receptors that inhibit glutamate release are only observed in the nerve terminals from young rats (one to three weeks), while the facilitatory effects are better seen in latter developmental stages (three to four weeks) and adult (two to three months) rats, coinciding with the development of the maximal capacity of glutamate uptake. These results indicate the existence of important developmental changes in the presynaptic control of glutamate release.