Mirian Silva do Carmo

SPM-1-Producing Pseudomonas aeruginosa: Analysis of the Ancestor Relationship Using Multilocus Sequence Typing, Pulsed-Field Gel Electrophoresis, and Automated Ribotyping

In Brazil, the spread of an endemic clone of SPM-1-producing Pseudomonas aeruginosa has been reported. Recently, a higher genomic variety has been observed among the SPM-1-producing P. aeruginosa isolates. The principal aim of this study was to analyze through multilocus sequence typing (MLST) analysis whether the recently isolated SPM-1-producing P. aeruginosa descend or not from a common ancestor. A total of 50 SPM-1-producing P. aeruginosa exhibiting 11 distinct ribotyping genotypes collected from 11 different Brazilian cities were studied. Three IMP-1-producing P. aeruginosa and two non-metallo-beta-lactamase-producing P. aeruginosa isolates were included in the study as controls. For assignment of allelic numbers and subsequent determination of sequence type (ST), the obtained sequences were compared to existing sequences in the MLST database (www.pubmlst.org/paeruginosa). The eBURSTv3 software was used in this study for establishing the evolutionary relationship and phylogenetic analysis. A total of 5 different STs were identified among 55 P. aeruginosa isolates. All of the SPM-1-producing P. aeruginosa presented an identical allelic profile (ST277), except for one strain. The three IMP-1-producing P. aeruginosa strains were classified as belonging to the ST593, whereas the non-metallo-beta-lactamase-producing P. aeruginosa showed two new distinct STs, ST594 and ST595. Our study shows that SPM-1-producing P. aeruginosa isolates as well as the IMP producers evaluated in this study descend from a common ancestor.

Molecular Characterization of Serine-, Alanine-, and Proline-Rich Proteins of Trypanosoma cruzi and Their Possible Role in Host Cell Infection

ABSTRACT We previously reported the isolation of a novel protein gene family, termed SAP (serine-, alanine-, and proline-rich protein), from Trypanosoma cruzi. Aided by the availability of the completed genome sequence of T. cruzi, we have now identified 39 full-length sequences of SAP, six pseudogenes and four partial genes. SAPs share a central domain of about 55 amino acids and can be divided into four groups based on their amino (N)- and carboxy (C)-terminal sequences. Some SAPs have conserved N- and C-terminal domains encoding a signal peptide and a glycosylphosphatidylinositol anchor addition site, respectively. Analysis of the expression of SAPs in metacyclic trypomastigotes by two-dimensional electrophoresis and immunoblotting revealed that they are likely to be posttranslationally modified in vivo. We have also demonstrated that some SAPs are shed into the extracellular medium. The recombinant SAP exhibited an adhesive capacity toward mammalian cells, where binding was dose dependent and saturable, indicating a possible ligand-receptor interaction. SAP triggered the host cell Ca2+ response required for parasite internalization. A cell invasion assay performed in the presence of SAP showed inhibition of internalization of the metacyclic forms of the CL strain. Taken together, these results show that SAP is involved in the invasion of mammalian cells by metacyclic trypomastigotes, and they confirm the hypothesis that infective trypomastigotes exploit an arsenal of surface glycoproteins and shed proteins to induce signaling events required for their internalization.

First description of blaCTX-M-14- and blaCTX-M-15-producing Escherichia coli isolates in Brazil.

We evaluated the antimicrobial resistance patterns and molecular characteristics of 11 extraintestinal Escherichia coli strains and 1 intestinal E. coli from human infections collected in Brazil. Two E. coli strains were nonsusceptible to extended spectrum cephalosporins (cefotaxime, ceftazidime, and cefepime); one isolated from diarrhea carried bla(CTX-M-14) and bla(TEM-1), whereas the other, isolated from tracheal secretion, carried bla(CTX-M-15) and bla(OXA-1). Five E. coli strains showed resistance to quinolones. Integrase associated with class 1 integron (intl1) was detected in 8 of the 12 E. coli strains belonging to various serotypes and this gene was carried by plasmids showing similar size. Pulsed-field gel electrophoresis showed that E. coli strains were genetically diverse, and phylogenetic grouping showed that the E. coli strains belonged to groups A, B2, and D (33.3%), respectively. This is the first report of E. coli isolates carrying bla(CTX-M-14) and bla(CTX-M-15) in Brazil. The presence of mobile elements containing antimicrobial resistance genes is worrisome since it could promote the dissemination of resistance and lead to the acquisition of resistance to other antimicrobials agents such as the carbapenems.

New multilocus sequence typing of MRSA in São Paulo, Brazil.

An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

Community-acquired methicillin-resistant Staphylococcus aureus carrying SCCmec type IV in southern Brazil

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen commonly associated with nosocomial infections. However, it has also been associated with community-acquired skin and soft tissue infections (CA-MRSA). There are few data on the identification and prevalence of CA-MRSA infections in Brazil. METHODS This is a cross-sectional study of 104 patients with community-acquired skin infections attending two health care centers in Porto Alegre, southern Brazil. MRSA isolates were characterized by molecular methods, including detection of the mecA gene by PCR, gene SCCmec typing, Panton-Valentine leukocidin (PVL) detection, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). RESULTS From the 104 samples, 58 Staphylococcus aureus isolates were obtained, of which five (8.6%) had a CA-MRSA-resistant profile. All five isolates had the mecA gene and amplified to SCCmec type IV. Analysis of chromosomal DNA by PFGE revealed the presence of two clusters related to international clones (OSPC and USA 300), with a Dice similarity coefficient >80%. The study was complemented by MLST, which detected three different strains: ST30, ST8, and ST45, the latter not presenting any relation with the clones compared in PFGE. CONCLUSIONS The presence of CA-MRSA reveals an important change in the epidemiology of this pathogen and adds new elements to the knowledge of the molecular biology of infections by MRSA with SCCmec type IV in southern Brazil.

Sepse por Staphylococus aureus resistente à meticilina adquirida na comunidade no sul do Brasil

Methicillin-resistant Staphylococcus aureus was initially described as a typical microorganism acquired in nosocomial infections. However, over recent years, community-acquired methicillin-resistant Staphylococcus aureus has been a cause of skin and soft-tissue infections. Serious infections such as pneumonia and sepsis can also occur. This report describes a case of sepsis in a child that was complicated by pneumonia secondary to soft tissue lesions that were due to community-acquired methicillin-resistant Staphylococcus aureus in southern Brazil. The patient was attended at the Emergency Unit with a history of injury caused by lower-limb trauma that evolved to cellulitis, pneumonia and sepsis.

Isolation and characterisation of genomic and cDNA clones coding for a serine-, alanine-, and proline-rich protein of Trypanosoma cruzi.

We report here the isolation and characterisation of genomic and cDNA clones encoding a Serine-, Alanine-, and Proline-rich protein (SAP) of Trypanosoma cruzi metacyclic trypomastigotes. The deduced peptides translated from these clones were characterised by a high content of residues of alanine, proline, serine, glycine, valine, and threonine distributed in several repeats: P(2-4), S(2-3), A(2-3), AS, SA, PA, AP, SP, PS, and TP. The repeats are partially homologous to the serine-, alanine-, and proline-containing motifs of Leishmania major and Leishmania mexicana proteophosphoglycans. Genes coding for SAP are part of a polymorphic family whose members are linked to members of gp85/sialidase and mucin-like gene families. This is consistent with the hypothesis that this genetic organisation could be a means by which T. cruzi co-ordinates the expression of major surface proteins.

Organization and expression of a multigene family encoding the surface glycoproteins of Trypanosoma cruzi metacyclic trypomastigotes involved in the cell invasion

Departamento de Micro, Imuno e Parasitologia, Escola Paulista de Medicina, Unifesp, Rua Botucatu 862, 04023-062 Sao Paulo, SP, Brasil *Unidad de Parasitologia, Universidad de Antofagasta, Av. Coloso s/n, Casilla 170,Antofagasta, Chile **Instituto de Biologia Experimental, Universidad Central de Venezuela, Aptado 47525,Caracas 1041-A, VenezuelaKey words: Trypanosoma cruzi - metacyclic trypomastigotes - surface glycoprotein genes - genomic organization- transcription

Structure and transcription of genes encoding the surface glycoprotein antigens gp90 and gp82 of Trypanosoma cruzi metacyclic trypomastigotes

SUMMARY OF COMMUNICATIONS CELL DAMAGE AND NEUROGENESIS IN THEDENTATE GRANULE CELL LAYER OF ADULT RATSAFTER PILOCARPINE- OR KAINATE-INDUCEDSTATUS EPILEPTICUS CovolanL.,RibeiroL.T.C.,LongoB.M.andMelloL.E.A.M. Department of Physiology, UNIFESP, 04023-900 Sao Paulo,SP.Presented by A.C.M.Paiva Dentate granule cells are generally considered to berelatively resistant to excitotoxicity and have been asso-ciated to robust synaptogenesis after neuronal damage.Synaptic reorganization of dentate granule cell axons,the mossy fibers, has been suggested to be relevant forhyperexcitability in human temporal lobe epilepsy andanimal models. A recent hypothesis has suggested thatmossy fiber sprouting is dependent on newly formed den-tate granule cells. However, we have recently demon-strated that cycloheximide (CHX) can block the mossyfiber sprouting that would be otherwise induced by dif-ferent epileptogenic agents and do not interfere withepileptogenesis in those models. Here, we investigatedcell damage and neurogenesis in the dentate gyrus ofpilocarpine- or kainate-treated animals with or withoutthe co-administration of CHX. Dentate granule cells werehighly vulnerable to pilocarpine induced-status epilep-ticus (SE), but hardly damaged by kainate induced-SE.CHX-pretreatment markedly reduced the number of in-jured neurons after pilocarpine-induced SE. Induction ofSE dramatically increased the mitotic rate of KA and KA+ CHX treated animals. Induction of SE in animals in-jected with pilocarpine alone led to increases of betweentwo to sevenfold in the mitotic rate of dentate granulecells as compared to increases of between five and thirty-fold for pilocarpine+CHX animals. These observationsindicate that in presence of cycloheximide the increase ofthe mitotic rate after pilocarpine-induced SE may be duetoprotectionofavulnerableprecursorcellpopulationthatwould otherwise degenerate. We further suggest that themossy fiber sprouting and neurogenesis of granule cellsare not necessarily related events. — ( September 14,1999 ).