Antimicrobial susceptibility of air-dispersed microorganisms in dental settings.

To determine the number and the susceptibility of microorganisms collected in a clinical environment against the antimicrobial agents used commonly in dentistry, petri dishes containing trypticase soy agar were exposed to air in different sites of a multi-chair dental clinic before, during, and after multiple clinical procedures and incubated for 24 hours under aerobic conditions. Colonies were identified by Gram stain technique and biochemical tests. Commercial paper disks containing widely prescribed antimicrobial agents (beta-lactams, macrolides, and clindamycin) were used to perform the antimicrobial susceptibility tests. The groups (colony forming units = cfu/m2/min) were submitted to the Kruskal-Wallis test (alpha = 5.0%), considering different clinical situations and environmental sites. During clinical procedures, the number of microorganisms increased (p < 0.05). This study highlights the need for established strategies to prevent resistant bacterial strains from emerging in dental settings.

Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA.

Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content. All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp. The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established. Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition. The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil.

The electrophoretic profiles of outer membrane proteins and lipopolysaccharide of sixty-five enterotoxigenic Escherichia coli of different serotypes and virulence-associated factors, toxin and colonization factors were determined. A close relationship between serotype and outer membrane protein and lipopolysaccharide patterns could be observed. No correlation could be found between the electrophoretic profiles and the expression of virulence-associated factors. The observed homogeneity of outer membrane protein and lipopolysaccharide profiles suggested the presence of only a few clones in the samples studied, and supported the use of outer membrane protein and lipopolysaccharide analysis as a useful epidemiological tool in the characterization of enterotoxigenic Escherichia coli isolates.

Clonal relationships among Escherichia coli serogroup O6 isolates based on RAPD.

The genetic diversity in a group of Escherichia coli strains belonging to serogroup O6 but expressing different H antigens was investigated by random amplification of polymorphic DNA (RAPD). Isolates of serotypes H16, H1, H31, and non-motile (NM) strains were typed using a set of 3 primers with different G + C contents. The amplified band arrays allowed the identification of 3 main clonal clusters corresponding to each O:H serotype analyzed. Based on their RAPD profiles NM strains could be assigned to either H1 or H31 serotypes. The results indicate that the flagellar antigen and the RAPD fingerprint represent reliable clonal markers in this E. coli group.

Identification of yebG as a DNA damage-inducible Escherichia coli gene.

The nucleotide sequence upstream of the Escherichia coli yebG gene presents features similar to those found in SOS system regulatory sites (putative SOS box, -10 and -35 promoter boxes and a ribosome binding site). Operon fusion assays demonstrate now that this region controls transcription in a recA-, lexA-dependent way and that the reporter gene expression is inducible by DNA damage consequent to mitomycin C treatment. Increased expression does not result from an increase in plasmid copy number. These results indicate that yebG is a novel SOS regulon gene. The yebG product is predicted to be a 96 amino acid residue, 10.7 kDa protein whose function is not yet known. Unlike other SOS genes, the construct carrying the yebG regulatory region is not stationary phase inducible.

Comparison of outer membrane protein and lipopolysaccharide profiles of enterotoxigenic Escherichia coli strains isolated in São Paulo, Brazil.

The outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns of 12 strains of serogroups of enterotoxigenic E. coli frequently isolated in São Paulo city were determined by fractionation techniques and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Five O6, three O78 and four O128 serogroup isolates of different serotypes (flagellar antigens) and virulence factors (toxins and colonization factor antigens) showed a high degree of variability in their OMP pattern and at least nine groups could be identified. The analysis of LPS patterns by SDS-PAGE showed a homogeneous profile for the O6 strains and some minor differences for the O128 and O78 strains. The present data indicate that analysis of OMP and LPS by SDS-PAGE may further improve the discriminating ability of extensively used serological techniques or the detection of virulence factors and could be a useful tool in epidemiological studies of enterotoxigenic E. coli (ETEC) strains from this area.

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity.

1. Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. 2. Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end. Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. 3. The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay. The full-length replicase is active. The aminoterminal truncated enzyme had 0.02% activity of the intact one. 4. This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli.

1. The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. 2. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10(5). The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha 32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. 3. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. 4. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli.

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to rebuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid.

[Aneurysmal bone cyst of the lumbar spine. Medico-surgical experience. A case report]. / Quiste óseo aneurismático de columna vertebral lumbar. Experiencia médico-quirúrgica. Reporte de un caso.

The aneurysmal bone cyst (ABC) of the lumbar spine is an infrequent entity, of undefined nature and aggressive behavior; there are only a few reports in Mexico, so we present the case of a patient with radicular and lumbar pain that progressed to pelvic limb paresthesia. Lesions in the vertebral body of L1 were located with imaging studies and confirmed with biopsy; treatment with corticosteroids was started and later resection of the lesion was performed together with stabilization of the spine using first a posterior approach and at a second stage an anterior approach. Corticosteroid therapy led to remission of tumor progression, so only vacuolated areas and destruction of the posterior wall were found, leading to instability and radiculopathy. This type of lesions usually appears in long bones and less often in the spine, but it is even rarer in the lumbar spine. The prognosis of ABC is usually good, with the exception of the rare cases of extremely disseminated cysts located in the spine. It is therefore important to consider this before deciding what the best approach is for each patient, as well as the appropriate medico-surgical measures.

Iron-dependent transcription of the frpB gene of Helicobacter pylori is controlled by the Fur repressor protein.

We have overexpressed and purified the Helicobacter pylori Fur protein and analyzed its interaction with the intergenic regions of divergent genes involved in iron uptake (frpB and ceuE) and oxygen radical detoxification (katA and tsaA). DNase I footprint analysis showed that Fur binds specifically to a high-affinity site overlapping the P(frpB) promoter and to low-affinity sites located upstream from promoters within both the frpB-katA and ceuE-tsaA intergenic regions. Construction of an isogenic fur mutant indicated that Fur regulates transcription from the P(frpB) promoter in response to iron. In contrast, no effect by either Fur or iron was observed for the other promoters.

Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans.

The genetic diversity of 47 enterotoxigenic Escherichia coli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD). Informative band arrays were obtained with three 10-mer primers with G+C contents of 50, 60, and 70%. Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established. The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil. Moreover, the RAPD technique revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed. These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.

Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 29 enterotoxigenic Escherichia coli strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis. The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT. Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common. These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group.

Beyond serotypes and virulence-associated factors: detection of genetic diversity among O153:H45 CFA/I heat-stable enterotoxigenic Escherichia coli strains.

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like São Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.

LexA repressor forms stable dimers in solution. The role of specific dna in tightening protein-protein interactions.

Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d). In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.

Comparison of outer membrane protein and lipopolysaccharide profiles of enterotoxigenic Escherichia coli strains isolet in Säo Paulo, Brazil

The outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns of 12 strains of serogroups of enterotoxigenic E. coli frequntly isolated in Säo Paulo city werte determined by fractionation techniques and by sodium dodecyl sulfate-plyacrylamide gel electrophoresis (SDS-PAGE). Five O6, three O78 and four O128 serogroup isolates of different serotypes (flagellar antigens) and virulence factors (toxins and colonization factor antigens) showed a high degree of variability in their OMP pattern and at least nine groups could be identified. The analysis of LPS aptterns by SDS-PAGE showed a homogenous profile for the O6 strains and some minor differences for the O128 and 078 strains. The oresented data indicate that analysis of OMP and LPS by SDS-PAGE may further improve the discriminating ability of extensively used serological techniques or the detection of virulence factors and could be a useful tool in epidemiological studies of enterotoxigenic E. coli (ETEC) strains from this area

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity

Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. Two constructions were developed to express either a full-lenghtt or truncated enzyme lacking the 20 aminoacids at the N-terminal en. Bacterial extr5acts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. The biological activity of these two molecules was tested using a poly(A)-dep[endent oligo(U)-primed poly(U)-polymer4ase assay. The full-lenght replicase is active. The aminoterminal truncated wnzyme had 0.02% activity o9f the intact5 one. This result indicates the importaqnce of the twenty N-terminal amino acids for the activity of FMDV RNA dependent RNMA polymerase

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli

The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid