Subtyping of Listeria monocytogenes isolates by actA gene sequencing, PCR-fingerprinting, and cell-invasion assay.

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.

The impact of environmental stress on Listeria monocytogenes virulence.

Listeria monocytogenes, a significant food-borne pathogen, must defy a variety of conditions encountered in the food environment and during the infection process. In reaction to adverse conditions, the bacteria significantly change their metabolism, inducing a stress response which is mediated by a range of alternative sigma factors. The extent of the response to stress was shown to vary in the L. monocytogenes population. According to recent evidence a major L. monocytogenes alternative sigma factor, designated sigma B (sigma B), regulates some virulence genes in response to stress, which supports an older hypothesis that stress-resistant strains should be more pathogenic. The induction of sigma B-dependent genes may also be important from the point of view of food hygiene. It seems that stress response activation can paradoxically enhance resistance to agents used in food preservation. Therefore, monitoring the expression of sigma B-dependent genes can serve as a useful marker to assess the innate resistance of L. monocytogenes strains. This knowledge will allow the design of new methods with sequential preservation steps that could inactivate the bacteria without inducing their stress response.

Importance of newly detected staphylococcal enterotoxins for food safety.

Staphylococcal enterotoxins (SEs) are emetic toxins causing food poisoning in humans, because of their biological activity and structural relatedness They have been classified as members of the pyrogenic exotoxin superantigen family Among them nine major antigenic types of emetic enterotoxins were recognized In recent years several newly detected SEs were also discriminated, but their occurrence and role in human and animal diseases are not fully understood Neverthless, evidences of their pathogenic role and broad distribution in staphylococcal strains cumulate Therefore their importance as potential risk factor for food safety becomes essential For this reason their properties, genetic determinants and supposed mechanisms of the pathogenic activity are discussed in respect of their potential hazard to human health.

The profiles of enterotoxin genes in Staphylococcus aureus from nasal carriers.

AIMS: To evaluate the occurrence of enterotoxin genes in Staphylococcus aureus recovered from nasal carriers. METHODS AND RESULTS: Eighty S. aureus strains were tested for the presence of 17 new enterotoxin genes using multiplex-PCR. Sixty-one isolates were found to carry enterotoxin genes. The majority of the enterotoxigenic isolates carried enterotoxin gene cluster (egc) genes, namely seg, sei, sem, sen and seo. The egc type containing the seu gene was found in 19 of the 47 isolates with egc-like genes. Interestingly, no seu-containing egc coexisted with sec and sel, as was the case for a considerable portion of the isolates carrying a seu-negative egc. The tst gene was detected in two isolates carrying sec and sel only and in eight isolates carrying seu, but not in the isolates containing the seu-negative egc type. CONCLUSIONS: The genes forming an egc were found to be predominant in S. aureus from nasal carriers. The coexistence of a seu-positive egc with tst in contrast to an egc lacking the seu gene apparently is not associated with the presence of tst and can reflect a difference between these gene groupings. SIGNIFICANCE AND IMPACT OF THE STUDY: The egc types carried by the analysed isolates seem to have an influence on the distribution of other genes located on staphylococcal pathogenicity islands, which may modulate the repertoire of virulence factors carried by a single S. aureus strain.

Superantigen types in Staphylococcus aureus isolated from patients with cystic fibrosis.

The screening of 17 SAg genes of S. aureus isolated from the sputum of cystic fibrosis (CF) patients revealed that among 47 genetically different strains, 39 (83 %) carried SAg genes. Superantigens forming enterotoxin gene cluster were detected in 20 strains. The 2nd most common superantigen type was selk detected in 13 strains. In 9 strains, selk occurred together with the sea gene. Out of 74 strains recovered from nasal carriers, 56 (75 %) were found to carry SAg genes, 38 carried egc genes, while selk was detected in 5 strains. The predominant SAg types in both investigated S. aureus populations were egc and selk/sea, but selk gene frequency was significantly higher in the CF-derived strains.

Occurrence of enterotoxigenic strains of Staphylococcus aureus in raw poultry meat.

The aim of this work was to determine the contamination of raw poultry meat with enterotoxigenic strains of S. aureus, using the PCR method. PCR is a rapid and sensitive method, which can show the presence in food of enterotoxigenic strains of S. aureus on the basis of specific gene sequences and detect the potential source of contamination before enterotoxins are produced. No coagulase-positive staphylococci strains were found in 65 samples of chicken parts, but these bacteria were present in 11 of 23 examined samples of minced turkey meat (48%). Using the primers for enterotoxin genes A to C, 4 of the 11 isolated S. aureus strains showed a positive result in the PCR. Three of the isolates represented the SEB gene and remaining one the SEC gene. The results obtained showed that PCR is sensitive and rapid method which may be used for detection and identification of enterotoxigenic S. aureus.

Structure of 5-hydroxy-2-hydroxymethyl-1-methylpyrid-4-one and 13C NMR relaxation studies of its gadolinium(III) complex.

In order to further elucidate the properties and biological behavior of 5-hydroxy-2-hydroxymethyl-1-methylpyrid-4-one (M1), its X-ray structure has been determined, and the ability of its gadolinium complex to enhance the relaxation of 13C nuclei has been examined. X-ray analysis using Mo K alpha radiation shows that M1 crystallizes in the monoclinic space group C2/c with a complex intermolecular array of hydrogen bonding. No water molecules were present within the unit cell. Gd(M1)2NO3 x 3H2O has been prepared and found to be very soluble in water. The effect of low concentrations of Gd(III) on enhancing the 13C relaxation times of M1 was examined. Trace amounts of Gd(NO3)3 x 6H2O resulted in significant decreases in the relaxation time of certain carbon atoms relative to the control measurements, and these data indicate that carbon atoms which bear donor atoms for Gd(III) undergo a significantly greater relaxation than the other carbons. The water solubility and hydrophilic character of this complex suggest that it may prove useful for the determination of metal binding sites on peptides and oligonucleotides.

Programmed instruction: biotherapy, module IV. interleukins.

This is the fourth in a series of five self-learning modules reviewing biotherapy. The focus of this module is the interleukins, biological response modifiers utilized in the treatment of some cancers.

Chelating agent inhibition of Trypanosoma cruzi epimastigotes in vitro.

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 micrograms/mL. The most effective compounds included N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included D-penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 microgram/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interfere with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.

Mobilization of iron by chiral and achiral anionic 3-hydroxypyrid-4-ones.

In the search for 3-hydroxypyrid-4-ones with enhanced iron-mobilizing ability, seven chiral, anionic amino acid derivatives of maltol (3-hydroxy-2-methyl-4-pyrone) have been synthesized, utilizing L-methionine, L-serine, L-leucine, L-phenylalanine, L-glutamic acid, and the D- and L-isomers of alanine. Two achiral, aromatic compounds were also synthesized and compared with the phenylalanine derivative. The biliary iron excretion following iv injection and the urinary iron excretion following po administration were measured using female Sprague-Dawley rats and compared to that of the standard, 1,2-dimethyl-3-hydroxypyrid-4-one (L1). While none of the compounds was as effective as L1 in enhancing the urinary excretion of iron, all monoanionic chelators increased excretion relative to the controls. All monoanionic compounds were at least equivalent to L1 in enhancing the biliary excretion of iron, with the methionine, leucine, and benzoate derivatives surpassing the standard and the other aromatic compounds also showing strong activity. The dianionic glutamate derivative showed low activity relative to the controls for both urinary and biliary iron excretion. No significant difference in iron excretion was observed due to variation in chirality; molecular weight and the number of negative charges appeared to have the greatest influence on the ability of the various derivatives to enhance iron excretion. In order to evaluate the relative purity of the stereoisomers, the alanine derivatives were analyzed by circular dichroism. Further characterization was provided by UV/vis spectroscopy for all compounds and X-ray crystallography for the novel dianionic derivative.

Structure/activity relationships affecting the ability of monoanionic 3-hydroxyprid-4-ones to mobilize iron.

Current attempts to remove iron from individuals suffering from iron overload have encountered difficulty due to the toxicity of the administered chelating agent. In a search for iron chelators of potentially reduced toxicity, nine monoanionic compounds have been examined. To determine the chemical features which govern their ability to induce the excretion of iron, the compounds were administered to female Sprague-Dawley rats. All carboxylate derivatives were tested for biliary excretion following iv injection, as well as for urinary excretion following iv or po injection. Sulfonate derivatives were tested for biliary and urinary excretion as well, but only one representative compound was tested po. The biological activity of the new pyridinones was compared to that of 1,2-dimethyl-3-hydroxypyrid-4-one, L1, which served as the standard. While none of the chelators was able to surpass L1 in both urinary and biliary iron excretion, all of the chelators at least equaled L1 in one of these two areas following iv administration. Two derivatives surpassed the standard in mobilizing iron into the bile, and all others were statistically equivalent. In terms of urinary excretion, two compounds were equivalent to L1 after iv administration, although none of the compounds equaled L1 when administered orally. The structure of (1,4-dihydro-3-hydroxy-2-methyl-4-oxo-1- pyridyl)methanecarboxylic acid was determined by X-ray diffraction, as this compound showed higher activity than previously reported by other investigators. We speculate that these chelators utilize organ-specific, monoanionic transport systems in the liver and kidneys to mobilize iron and that their toxicity may be substantially less than that of their neutral analogs.

Synthesis and iron(III) binding properties of 3-hydroxypyrid-4-ones derived from kojic acid.

In an attempt to reduce the toxicity of the 3-hydroxypyrid-4-ones, the more hydrophilic derivatives of kojic acid were explored and compared to the standard, 1,2-dimethyl-3-hydroxypyrid-4-one, L1. The synthesis and iron(III) binding properties of these chelators are described. Neither these compounds nor the clinically effective 1,2-dimethyl-3-hydroxypyrid-4 one is able to completely remove all of the iron(III) from the Fe(III)EDTA complex in sodium acetate buffered solutions, when the 3-hydroxypyrid-4-one: Fe(III) ratio is 6:1. The ability of these compounds to enhance the urinary excretion of iron in rats indicates that the behavior of the 3-hydroxypyrid-4-ones derived from kojic acid is comparable to the analogous derivatives of maltol and ethyl maltol. The structure of the iron(III) complex of 3-hydroxy-6-hydroxymethyl-1-methylpyrid-4-one was determined by x-ray diffraction and found to be similar to the previously reported structure of the iron(III) complex of L1.

Rabies enzootic situation in animals on Lower Silesia in 1983-1992.

Considerable increase (average 70%) of rabies cases in animals on Lower Silesia, occurred in second half of the decade 1983-1992. Foxes were a reservoir of the virus and epizootic spread of the disease had been noted among them. In other animal species, both wildlife and domestic, only sporadic outbreaks were noted. They were a consequence of contact of these animals with infected foxes, in which from 85% to 90% incidences a year were stated. Parallel rise of rabies cases in cats makes this animal the most important potential vector of the disease to man.

Enhancement of iron excretion via monoanionic 3-hydroxypyrid-4-ones.

The ability of 3-hydroxypyrid-4-ones bearing either a carboxylic acid or sulfonic acid group to mobilize iron into the bile and urine of normal rats has been examined and compared with that produced by 1,2-dimethyl-3-hydroxypyrid-4-one (L1). The compounds tested were 3-hydroxy-1-methyl-4-oxopyridine-6-carboxylic acid and 1-[3-hydroxy-6-(hydroxymethyl)-4-oxopyridyl]-2-ethanesulfonic acid, whose synthesis, biological activity, and X-ray crystallographic properties are described. Although estimates of activity, based on polarity and membrane permeability, predict such compounds to be ineffective, they were found to have an iron-mobilizing ability similar to that of the compounds which do not bear any charge at physiological pH when given parenterally. When given orally, the 3-hydroxypyrid-4-one containing a carboxylate group enhanced the urinary excretion of iron, while the sulfonate analog did not substantially increase the excretion of iron in the urine relative to the controls. The results obtained here suggest that the previous emphasis on the preparation of 3-hydroxypyrid-4-ones that are electrically neutral at physiological pH is unnecessarily restrictive and that the presence of an appropriate group bearing a single negative charge is consistent with a high level of activity. It is proposed that such negatively charged molecules may gain access to the interior of cells in both the kidney and the liver via monoanionic transport systems. Such compounds may prove to be less toxic than the neutral 3-hydroxypyrid-4-ones.