Luis C. Patrito

Identification of a Novel Methicillin-Resistant Staphylococcus aureus Epidemic Clone in Córdoba, Argentina, Involved in Nosocomial Infections

ABSTRACT Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.

Molecular cloning and expression of the β-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.

A novel activity for a group of sesquiterpene lactones: inhibition of aromatase.

A group of eleven sesquiterpene lactones isolated from different Asteraceae species from north‐western Argentina were investigated for their inhibitory action on the estrogen biosynthesis. Seven of them, of different skeleton types, were found to inhibit the aromatase enzyme activity in human placental microsomes, showing IC50 values ranging from 7 to 110 μM. The most active were the guaianolides 10‐epi‐8‐deoxycumambrin B (compound 1), dehydroleucodin (compound 2) and ludartin (compound 3). These compounds were competitive inhibitors with an apparent K i=4 μM, K i=21 μM and K i=23 μM, respectively. Compounds 1 and 2 acted as type II ligands to the heme iron present in the active site of aromatase cytochrome P450 (P450arom). Besides, all of them failed to affect the cholesterol side‐chain cleavage enzyme activity on human placental mitochondrias. This is the first report on the aromatase inhibitory activity of this group of natural compounds.

The 59-kDa polypeptide constituent of 8–10-nm cytoplasmic filaments in Neurospora crassa is a pyruvate decarboxylase

The fungus Neurospora crassa harbors large amounts of cytoplasmic filaments which are homopolymers of a 59-kDa polypeptide (P59Nc). We have used molecular cloning, sequencing and enzyme activity measurement strategies to demonstrate that these filaments are made of pyruvate decarboxylase (PDC, EC 4.1.1.1), which is the key enzyme in the glycolytic-fermentative pathway of ethanol production in fungi, and in certain plants and bacteria. Immunofluorescence analyses of 8-10-nm filaments, as well as quantitative Northern blot studies of P59Nc mRNA and measurements of PDC activity, showed that the presence and abundance of PDC filaments depends on the metabolic growth conditions of the cells. These findings may be of relevance to the biology of ethanol production by fungi, and may shed light on the nature and variable presence of filament bundles described in fungal cells.

Regulation of the 3β-hydroxysteroid dehydrogenase activity in tissue fragments and microsomes from human term placenta: Kinetic analysis and inhibition by steroids

The effects of 50 microM of progesterone (P4), estradiol (E2), estrone (E1), estriol (E3), dehydroepiandrosterone (DHIA), androstenedione (delta 4) and testosterone (T) on the bioconversion of [3H]pregnenolone (6 nM) to [3H]P4 were investigated by incubating 200 mg of tissue fragments as well as equivalent aliquots of microsomes from human term placenta during 30 min. All the steroids assayed, except E3, significantly inhibited the [3H]P4 formation in a microsome incubation system with respect to the control assay (P less than 0.001). Conversely in a tissue incubation system. P4, E1 as well as E3 had no effect on [3H]pregnenolone bioconversion while E2 slightly decreased the [3H]P4 formation (P less than 0.05) compared with the control. A significant inhibition was observed in this system with the other steroids (P less than 0.001). To investigate these apparent different results of inhibition-noninhibition of the same steroids irrespective of the system of incubation used, the effects of P4, E2 and T on 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activity were studied in tissue fragments and microsomes in kinetic terms. The results found indicate that these steroids inhibited in a competitive fashion the 3 beta-HSD activity in both systems. The different Ki values found in tissue fragments and microsomes respectively for P4 (1.8 microM vs 0.5 microM), E2 (2.3 microM vs 0.6 microM) and T (0.25 microM vs 0.3 microM) explain the bioconversion results obtained in presence of 50 microM of the same steroids. These results include inhibition of [3H]P4 formation by T in tissue fragments as well as in microsomes whereas P4 and E2 inhibited the [3H]P4 formation only in microsomes. Furthermore, the comparison of these Ki values with the available data of intraplacental and circulating concentrations of the same steroids in human term pregnancy suggest that only P4 would be expected to cause marked 3 beta-HSD inhibition in physiological conditions.

Nucleotide sequence of a pregnancy-specific β1 glycoprotein gene family member

The pregnancy-specific β1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5′ flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5′ further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.

Transcriptional Control of the Human Pregnancy-specific Glycoprotein 5 Gene is Dependent on Two GT-boxes Recognized by the Ubiquitous Specificity Protein 1 (Sp1) Transcription Factor

Pregnancy-specific glycoprotein 5 gene (PSG-5) belongs to the human pregnancy-specific glycoprotein family, encoded by eleven highly similar and transcriptionally active genes. High levels of PSG biosynthesis are restricted to the placenta syncytiotrophoblast and are essential for the maintenance of normal gestation in mammalian species. We have investigated here the nature of the transcription factors that recognize the FP1 (-455/-433) and the CPE (-147/-140) regulatory sequences that significantly contribute to basal PSG-5 promoter activity. Both elements bear a similar GT-box motif; and DNA-protein complex formation, as well as promoter activity, is largely dependent on the integrity of these GT-box sequences. Gel shift, super gel shift and UV-crosslinking experiments clearly demonstrate that the ubiquitous specificity protein 1 (Sp1) is the major transcription factor involved in complex formation with both cis-acting elements in normal term placenta tissue and in PSG-non-expressing COS-7 cells. Furthermore, transfection experiments indicate that Sp1 activates PSG-5 promoter constructs. In addition, we show that Sp1 is indeed co-expressed with PSG genes in the syncytiotrophoblast cells, stressing its potential role in the in vivo regulation of PSG expression.

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron.

The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46.5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46.5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.

Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

Effect of estradiol-17β on the conversion of pregnenolone to progesterone in human term placenta incubated in vitro

The effect of different doses of estradiol-17 beta (E2) on the metabolic pregnenolone to progesterone pathway in fragments of human term placenta incubated in vitro was studied. Doses considered as being physiological of 0.09 and 0.9 microM had a stimulatory effect on the conversion (p less than 0.008 to 0.016). However, a supraphysiological dose of 45 microM showed an inhibitory activity related to the maximal stimulation (p less than 0.03). A dose of 0.9 microM E2 favoured the accumulation of (3H)-progesterone in the tissue (p less than 0.05). These results suggest that E2 may regulate the synthesis of progesterone in human term placenta.