Case Series Study of Melioidosis, Colombia.

We report 7 cases of melioidosis in Colombia and comparision of 4 commercial systems for identifying Burkholderia pseudomallei. Phoenix systems were not a definitive method for identifying B. pseudomallei. For accurate identification, we recommend including this bacterium in the library databases of matrix-assisted laser desorption/ionization mass spectrometry systems in Latin America.

Multiparameter flow cytometry analysis of leukocyte markers for diagnosis in preterm neonatal sepsis.

PURPOSE: Neonatal sepsis is an important public health concern worldwide due to its immediate lethality and long-term morbidity rates, Clinical evaluation and laboratory analyses are indispensable for diagnosis of neonatal sepsis. However, assessing multiple biomarkers in neonates is difficult due to limited blood availability. The aim is to investigate if the neonatal sepsis in preterm could be identified by multiparameter analysis with flow cytometry. MATERIALS AND METHODS: The expression of activation-related molecules was evaluated by flow cytometry in newborn with or without risk factors for sepsis. RESULTS: Our analysis revealed that several markers could be useful for sepsis diagnosis, such as CD45RA, CD45RO, or CD71 on T cells; HLA-DR on NKT or classic monocytes, and TREM-1 on non-classic monocytes or neutrophils. However, ROC analysis shows that the expression of CD45RO on T lymphocytes is the only useful biomarker for diagnosis of neonatal late-onset sepsis. Also, decision tree analyses showed that CD45RO plus CD27 could help differentiate the preterm septic neonates from those with risk factors. CONCLUSIONS: Our study shows a complementary and practical strategy for biomarker assessment in neonatal sepsis.

Transcriptome analysis of genes controlled by luxS/autoinducer-2 in Salmonella enterica serovar Typhimurium.

The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.

Rapid concentration of Bacillus and Clostridium spores from large volumes of milk, using continuous flow centrifugation.

Deliberate or accidental contamination of foods such as milk, soft drinks, and drinking water with infectious agents or toxins is a major concern to health authorities. There is a critical need to develop technologies that can rapidly and efficiently separate and concentrate biothreat agents from food matrices. A key limitation of current centrifugation and filtration technologies is that they are batch processes with extensive hands-on involvement and processing times. The objective of our studies was to evaluate the continuous flow centrifugation (CFC) technique for the rapid separation and concentration of bacterial spores from large volumes of milk. We determined the effectiveness of the CFC technology for concentrating approximately 10(3) bacterial spores in 3.7 liters (1 gal) of whole milk and skim milk, using Bacillus subtilis, Bacillus atrophaeus, and Clostridium sporogenes spores as surrogates for biothreat agents. The spores in the concentrated samples were enumerated by using standard plating techniques. Three independent experiments were performed at 10,000 rpm and 0.7 liters/min flow rate. The mean B. subtilis spore recoveries were 71.3 and 56.5% in skim and whole milk, respectively, and those for B. atrophaeus were 55 and 59.3% in skim and whole milk, respectively. In contrast, mean C. sporogenes spore recoveries were 88.2 and 78.6% in skim and whole milk, respectively. The successful use of CFC to concentrate these bacterial spores from 3.7 liters of milk in 10 min shows promise for rapidly concentrating other spores from large volumes of milk.

Recovery and functional validation of hidden soil enzymes in metagenomic libraries.

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.

Grapefruit juice and its furocoumarins inhibits autoinducer signaling and biofilm formation in bacteria.

Cell-to-cell communications in bacteria mediated by small diffusible molecules termed as autoinducers (AI) are known to influence gene expression and pathogenicity. Oligopeptides and N-acylhomoserine lactones (AHL) are major AI molecules involved in intra-specific communication in gram-positive and gram-negative bacteria respectively, whereas boronated-diester molecules (AI-2) are involved in inter-specific communication among both gram-positive and gram-negative bacteria. Naturally occurring furocoumarins from grapefruit showed >95% inhibition of AI-1 and AI-2 activities based on the Vibrio harveyi based autoinducer bioassay. Grapefruit juice and furocoumarins also inhibited biofilm formation by Escherichia coli O157:H7, Salmonella typhimurium and Pseudomonas aeruginosa. These results suggest that grape fruit juice and furocoumarins could serve as a source to develop bacterial intervention strategies targeting microbial cell signaling processes.

Necrotizing fasciitis caused by Apophysomyces variabilis in an immunocompetent patient.

Mucormycosis caused by Apophysomyces variabilis is rarely reported in humans. A case of A. variabilis infection in an immunocompetent men after friction burns in a car accident is described. The infection presented as a rapidly progressive necrotizing infection of the skin and soft tissue, which required extensive surgical debridement and total colonic defunctioning colostomy associated with prolonged antifungal therapy. A. variabilis infection should be considered as a differential diagnosis of rapidly progressive necrotizing skin and soft tissue infections in immunocompetent individuals.