The role of virulence factors in the outcome of staphylococcal peritonitis in CAPD patients.

BACKGROUND: Peritonitis continues to be the most frequent cause of peritoneal dialysis (PD) failure, with an important impact on patient mortality. Gram-positive cocci such as Staphylococcus epidermidis, other coagulase-negative staphylococci (CoNS), and Staphylococcus aureus are the most frequent etiological agents of PD-associated peritonitis worldwide. The objective of the present study was to compare peritonitis caused by S. aureus and CoNS and to evaluate the factors influencing outcome. METHODS: Records of 86 new episodes of staphylococcal peritonitis that occurred between 1996 and 2000 in the Dialysis unit of a single university hospital were studied (35 due to S. aureus, 24 to S. epidermidis and 27 to other CoNS). The production of slime, lipase, lecithinase, nuclease (DNAse), thermonuclease (TNAse), alpha- and beta-hemolysin, enterotoxins (SEA, SEB, SEC, SED) and toxic shock syndrome toxin-1 (TSST-1) was studied in S. aureus and CoNS. Antimicrobial susceptibility was evaluated based on the minimal inhibitory concentration determined by the E-test. Outcome predictors were evaluated by two logistic regression models. RESULTS: The oxacillin susceptibility rate was 85.7% for S. aureus, 41.6% for S. epidermidis, and 51.8% for other CoNS (p = 0.001). Production of toxins and enzymes, except for enterotoxin A and alpha-hemolysin, was associated with S. aureus episodes (p < 0.001), whereas slime production was positive in 23.5% of CoNS and 8.6% of S. aureus strains (p = 0.0047). The first model did not include enzymes and toxins due to their association with S. aureus. The odds of resolution were 9.5 times higher for S. epidermidis than for S. aureus (p = 0.02) episodes, and were similar for S. epidermidis and other CoNS (p = 0.8). The resolution odds were 68 times higher for non-slime producers (p = 0.001) and were not influenced by oxacillin resistance among vancomycin-treated cases (p = 0.89). In the second model, the resolution rate was similar for S. aureus and S. epidermidis (p = 0.70), and slime (p = 0.001) and alpha-hemolysin (p = 0.04) production were independent predictors of non-resolution. CONCLUSION: Bacterial species and virulence factors rather than antibiotic resistance influence the outcome of staphylococcal peritonitis.

Transglutaminase 2 inhibitor, KCC009, disrupts fibronectin assembly in the extracellular matrix and sensitizes orthotopic glioblastomas to chemotherapy.

Transglutaminase 2 (TG2, a.k.a. tissue transglutaminase) belongs to a family of transglutaminase enzymes that stabilize proteins by affecting covalent crosslinking via formation of amide bonds. Cell surface TG2 is directly involved as an adhesive receptor in cell-extracellular matrix (ECM) interactions. Here, we show that TG2 activity is elevated in glioblastomas compared with non-neoplastic brain. Immunofluorescent studies showed increased staining of fibronectin colocalized with TG2 in the ECM in glioblastomas. In addition, small clusters of invading human glioblastoma cells present in non-neoplastic brain parenchyma secrete high levels of TG2 and fibronectin that distinguish them from normal brain stroma. Downregulation of TG2 in U87MG glioblastoma cells with RNAi demonstrated decreased assembly of fibronectin in the ECM. Treatment with KCC009 blocked the remodeling of fibronectin in the ECM in glioblastomas in both in vitro and in vivo studies. KCC009 treatment in mice harboring orthotopic glioblastomas (DBT-FG) sensitized the tumors to N,N'-bis(2-chloroethyl)-N-nitrosourea chemotherapy, as measured by reduced bioluminescence, increased apoptosis and prolonged survival. The ability of KCC009 to interfere with the permissive remodeling of fibronectin in the ECM in glioblastomas suggests a novel target to enhance sensitivity to chemotherapy directed not only at the tumor mass, but also invading glioblastoma cells.

Usefulness of catheter tip culture in the diagnosis of neonatal infections.

OBJECTIVE: To determine the number of colony-forming units (CFU) that best correlates with catheter-related infections (CRI) in newborns. METHODS: This was a prospective study of semiquantitative cultures of catheter tips obtained from newborns in the neonatal unit at Faculdade de Medicina de Botucatu, state of São Paulo, Brazil. The microorganisms isolated from catheter and peripheral blood cultures were identified and submitted to a drug susceptibility test. The optimal cutoff point was determined by the receiver operating characteristic (ROC) curve. RESULTS: A total of 85 catheters obtained from 63 newborns were studied. Staphylococcus epidermidis was the predominant species in the catheters (75%). Eight of 11 (72.7%) CRI episodes were associated with coagulase-negative staphylococci, six of which were of the S. epidermidis type. ROC curve analysis indicated that the optimal cutoff point for the diagnosis of CRI was 122 CFU. CONCLUSIONS: The cutoff point of 122 CFU correlated best with the diagnosis of CRI in newborns.

Use of alkaline phosphatase fusions to study protein secretion in Bacillus subtilis.

We have constructed a vector designed to facilitate the study of protein secretion in Bacillus subtilis. This vector is based on a translational fusion between the expression elements and signal sequence of Bacillus amyloliquefaciens alkaline protease and the mature coding sequence for Escherichia coli alkaline phosphatase (phoA). We show that export of alkaline phosphatase from B. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. The vector design facilitates the insertion of heterologous coding sequences between the signal and phoA to generate three-part translational fusions. Such phoA fusions are easily analyzed by monitoring alkaline phosphatase activity on agar plates or in culture supernatants or by immunological detection. Exploitation of this methodology, which has proven to be extremely useful in the study of protein secretion in E. coli, has a variety of applications for studying protein secretion in B. subtilis.

Duplication-translocations of tryptophan operon genes in Escherichia coli.

Mutants of Escherichia coli were selected in which a single mutational event had both relieved the polar effect of an early trpE mutation on trpB and simultaneously released the expression of trpB from tryptophan repression. The frequency at which these mutations appeared was roughly equal to the frequency of point mutations. In each of these mutants, the mutation increased the function of trpB and also increased the activity of some, but not all, of the other four tryptophan operon genes. Genetic analysis showed that the mutations were not located within the trp operon since in each case the parental trp operon could be recovered from the mutants. Each mutant was shown to carry a duplication of a trp operon segment translocated to a new position near the trp operon. Polarity is relieved since the trpB duplication-translocation is not in the same operon as the trpE polar mutation. The duplicated and translocated segments are fused to operons not regulated by tryptophan, so trpB function is no longer subject to tryptophan repression. The properties of the mutants indicate that the length of the duplicated segment and the position to which it is translocated differ in each of the seven mutants studied. The duplications are unstable, but the segregation pattern observed is not consistent with a single crossover model for segregation. That such duplication-translocation events generate a variety of new genetic arrangements at a frequency comparable with point mutations suggests they may play an important role in evolution.

Localization of two functions of the phosphoribosyl anthranilate transferase of Escherichia coli to distinct regions of the polypeptide chain.

The trpD gene specifies a polypeptide which has both glutamine amidotransferase and phosphoribosyl anthranilate (PRA) transferase activities. Deletions fusing segments of trpD to the gene preceding it in the operon, trpE, were selected in strains carrying various trpD point mutations. The selection procedure required both that a deletion enter trpE and that it restore the PRA transferase activity which the parent trpD point mutant lacked. Deletion mutants were found which had PRA transferase activity although the first third of trpD was deleted. The existence of the mutants proves that a terminal segment of trpD is sufficient to specify a polypeptide having PRA transferase activity. The location of the deletion end points on the genetic map of trpD defines the extent of the trpD segment required for PRA transferase activity. This segment did not overlap the initial region of trpD required to specify the glutamine amidotransferase function of the trpD polypeptide. These results support the hypothesis (M. Grieshaber and R. Bauerle, 1972; H. Zalkin and L. H. Hwang, 1971) that the bifunctional trpD polypeptide might have evolved by fusion of a gene specifying a glutamine amidotransferase with a gene directing PRA transferase synthesis.

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction.

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.

ESTs from the basidiomycete Schizophyllum commune grown on nitrogen-replete and nitrogen-limited media.

Lambda phage cDNA libraries were constructed using mRNAs from the basidiomycete Schizophyllum commune grown on media with high or low nitrogen concentrations. A total of 440 clones were sequenced, representing 373 distinct transcripts. Of these, 166 showed significant similarity to annotated genes in GenBank. Those that could be tentatively identified using BLAST searches were classified by function using the Gene Ontology (GO) database. Genes with products involved in cell-cycle processes were more frequent in the nitrogen-limited libraries, while genes with products involved in protein biosynthesis were more frequent in the nitrogen-replete library. Overall, clones showed much greater similarity to the one publicly available basidiomycete genome, Phanerochaete chrysosporium, than to any of the ascomycete genomes.

Utilidade da cultura da ponta de cateter no diagnóstico de infecção neonatal / Usefulness of catheter tip culture in the diagnosis of neonatal infections

OBJETIVO: Determinar o número de unidades formadoras de colônias que melhor correlaciona com a infecção relacionada a cateter em recém-nascidos. MÉTODOS: Este foi um estudo prospectivo de culturas semiquantitativas de pontas de cateteres de recém-nascidos da unidade neonatal da Faculdade de Medicina de Botucatu. Os microrganismos isolados de cateteres e hemoculturas periféricas foram identificados e submetidos ao teste de sensibilidade a drogas. O ponto de corte ótimo foi determinado pela curva receiver operating characteristic (ROC). RESULTADOS: Foram estudados 85 cateteres de 63 recém-nascidos. A espécie Staphylococcus epidermidis foi prevalente (75 por cento) nos cateteres. Dos 11 episódios de infecção diagnosticados, oito (72,7 por cento) foram associados aos estafilococos coagulase-negativa, dos quais seis pertenciam à espécie S. epidermidis. Pela curva ROC, o ponto de corte ótimo para o diagnóstico de infecção relacionada a cateter foi 122 unidades formadoras de colônias. CONCLUSÃO: O ponto de corte 122 unidades formadoras de colônias melhor se correlacionou com o diagnóstico de infecção relacionada a cateter em recém-nascidos.

OBJECTIVE: To determine the number of colony-forming units (CFU) that best correlates with catheter-related infections (CRI) in newborns. METHODS: This was a prospective study of semiquantitative cultures of catheter tips obtained from newborns in the neonatal unit at Faculdade de Medicina de Botucatu, state of São Paulo, Brazil. The microorganisms isolated from catheter and peripheral blood cultures were identified and submitted to a drug susceptibility test. The optimal cutoff point was determined by the receiver operating characteristic (ROC) curve. RESULTS: A total of 85 catheters obtained from 63 newborns were studied. Staphylococcus epidermidis was the predominant species in the catheters (75 percent). Eight of 11 (72.7 percent) CRI episodes were associated with coagulase-negative staphylococci, six of which were of the S. epidermidis type. ROC curve analysis indicated that the optimal cutoff point for the diagnosis of CRI was 122 CFU. CONCLUSION: The cutoff point of 122 CFU correlated best with the diagnosis of CRI in newborns.

Comparison between qualitative and semiquantitative catheter-tip cultures: laboratory diagnosis of catheter-related infection in newborns / Comparação entre culturas qualitativa e semiquantitativa de ponta de cateter: diagnóstico laboratorial de infecção relacionada a cateter em recém-nascidos

This prospective study evaluated semiquantitative and qualitative catheter-culture methods for diagnosis of catheter-related infection (CRI) in newborns. Catheter tips from newborns admitted to the Neonatal Unit of the University Hospital of the Botucatu Medical School, UNESP were included in the study. Catheter cultures were performed with both semiquantitative and qualitative techniques. For CRI diagnosis, microorganisms isolated from catheter cultures and from peripheral blood cultures were identified and submitted to agent susceptibility test. The gold standard was the certain CRI diagnosis when same microorganism (specie and profile of susceptibility to agents) was isolated from both catheter tips and peripheral blood culture. A total of 85 catheters from 63 newborns were included in the study. The semiquantitative culture method, despite presenting lower sensitivity (90 percent), showed higher specificity (71 percent) when compared to 100 percent of sensitivity and 60 percent of specificity in the qualitative method. The identification of the microorganisms obtained from the catheter cultures showed a prevalence of coagulase-negative staphylococci(CNS) species. The specie Staphylococcus epidermidis (77.5 percent) was the prevalent in the catheters with positive semiquantitative cultures. Among 11 episodes with CRI diagnosis, 8 (72.7 percent) were associated with CNS species, of which 6 were S. epidermidis. Two episodes of CRI by S. aureus and one by Candida parapsilosis were also detected. The semiquantitative catheter-culture method showed advantages for CRI diagnosis in newborns when compared to the conservative qualitative method.

Este estudo prospectivo avaliou os métodos semiquantitativo e qualitativo de cultura de cateter para o diagnóstico de infecção relacionada a cateter (IRC) em recém-nascidos (RN). Foram incluídas pontas de cateteres provenientes de recém-nascidos internados na Unidade Neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu, UNESP. Foram utilizadas as técnicas semiquantitativa e qualitativa de cultura de cateter. Para o diagnóstico de IRC, os microrganismos isolados das culturas de cateteres e de hemoculturas periféricas foram identificados e submetidos ao teste de sensibilidade a antimicrobianos. O padrão ouro correspondeu ao diagnóstico de certeza de IRC, com o isolamento do mesmo microrganismo (espécie e perfil de sensibilidade a antimicrobianos) isolado em hemocultura periférica. Foram estudados 85 cateteres provenientes de 63 RN. A cultura semiquantitativa, embora tenha apresentado menor sensibilidade (90 por cento), apresentou uma maior especificidade (71 por cento) em comparação à sensibilidade de 100 por cento e especificidade de 60 por cento encontradas na cultura qualitativa. Através da identificação dos microrganismos obtidos nas culturas de cateteres, observou-se uma predominância de espécies de Estafilococos coagulase-negativa (ECN). A espécie Staphylococcus epidermidis foi a prevalente (77,5 por cento) nos cateteres com culturas semiquantitativas positivas. Dos 11 episódios de IRC diagnosticados, 8 (72,7 por cento) foram associados a espécies de ECN, dos quais 6 eram da espécie S. epidermidis. Também foram detectados dois casos de IRC por S. aureus e um caso por Candida parapsilosis. O método de cultura semiquantitativo cateter apresentou vantagens para o diagnóstico de IRC em RN quando comparado com o método qualitativo tradicional.