A novel strictly anaerobic recovery and enrichment system incorporating lithium for detection of heat-injured Listeria monocytogenes in pasteurized milk containing background microflora.

Heat-injured cells of Listeria monocytogenes were recovered from heated raw milk containing noninjured Enterococcus faecium by combining a simple method for obtaining strict anaerobiosis with a novel enrichment broth, Penn State University broth (PSU broth). Strictly anaerobic conditions were rapidly achieved by adding 0.5 g of filter-sterilized cysteine per liter to PSU broth and then purging the preparation with N2 gas. Little resuscitation or growth occurred in strictly anaerobic PSU broth without lithium chloride because of overgrowth by E. faecium. The growth of E. faecium decreased dramatically with increasing LiCl concentration; LiCl concentrations of 8 and 10 g/liter were completely bacteriostatic. The mechanism of inhibition by LiCl appeared to involve competition with the divalent cations Ca2+ and Mg2+. Heat-injured L. monocytogenes consistently recovered and grew rapidly in strictly anaerobic PSU broth containing 4, 6, or 7 g of LiCl per liter. The use of strictly anaerobic PSU broth containing 7 g of LiCl per liter permitted detection of severely heat-injured L. monocytogenes in one simple recovery-enrichment step by eliminating oxygen toxicity and inhibiting the growth of background microflora, without preventing the resuscitation and subsequent growth of heat-injured L. monocytogenes. L. monocytogenes heated in raw milk at 62.8 degrees C for 10, 15, and 20 min could be consistently recovered from strictly anaerobic PSU broth enrichment cultures at 30 degrees C after 48, 96, and 144 h, respectively, and hence, use of PSU broth may result in better recovery of both injured and noninjured cells from foods than currently used U.S. Department of Agriculture and Food and Drug Administration preenrichment procedures.     

The combined use of 131I-labeled antibody and the hypoxic cytotoxin SR 4233 in vitro and in vivo.

Radioimmunotherapy is hindered by the slow penetration of antibody molecules into tumors. Cells that are poorly targeted by antibody, because of their distance from feeding blood vessels, receive the lowest radiation dose, and this problem is compounded if there are radioresistant hypoxic cells present. It would be desirable to combine radioimmunotherapy with an agent that is preferentially toxic to these cells. SR 4233 is a potent hypoxic cytotoxin, and it was combined with 131I-NR-LU-10 to treat LS174T human colon adenocarcinoma multicell spheroids and nude mouse xenografts for these studies. Under conditions of severe hypoxia (< 0.01% O2), 2 h of pretreatment or 18 h of simultaneous treatment with SR 4233 did not significantly enhance the effectiveness of 131I-NR-LU-10 in spheroids. However, under aerobic conditions with a 10% fraction of hypoxic cells, there was more toxicity than would be predicted from simple additivity. Xenografts treated with 131I-NR-LU-10 + SR 4233 had a growth delay that was significantly longer than that achieved with 131I-NR-LU-10 alone. In both spheroids and xenografts, combined treatment produced about 10 times more cell killing than 131I-NR-LU-10 alone. The lack of enhancement in spheroids under complete hypoxia suggests that SR 4233 does not sensitize hypoxic cells to radiation damage. The results with aerobic spheroids and in vivo, where a portion of the cells were hypoxic, could be explained by the targeting of different cell populations (hypoxic and aerobic) by each therapeutic modality. This effect should also be enhanced by reoxygenation and reestablishment of the hypoxic fraction during treatment, thus allowing more than the initially hypoxic fraction of cells to be killed by the SR 4233.     

Design and synthesis of selective keto-1,2,4-oxadiazole-based tryptase inhibitors

Using a scaleable, directed library approach based on orthogonally protected advanced intermediates, we have prepared a series of potent keto-1,2,4-oxadiazoles designed to explore the P(2) binding pocket of human mast cell tryptase, while building in a high degree of selectivity over human trypsin and other serine proteases.     

The transmembrane isoform of Plasmodium falciparum MAEBL is essential for the invasion of Anopheles salivary glands

Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54) sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies.     

Pseudomonas aeruginosa: study of antibiotic resistance and molecular typing in hospital infection cases in a neonatal intensive care unit from Rio de Janeiro City,Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.     

Synthesis of sterically crowded derivatives of anomeric pairs of D-glucose disaccharides

Derivatization of carbohydrates is of considerable interest since the derivatives can be used for structural studies in the field of mass spectrometry. We report here the synthesis of a series of sterically crowded derivatives of various linkage and stereo-isomeric glucose-glucose disaccharides with the impetus being to understand the effect of these derivatized groups on fragmentation of the glycosidic bond and the development of methodology for discernment of the anomeric configuration. The synthesis of per-alkylated (methyl, ethyl, propyl, butyl, and pentyl), per-esterified (acetyl, pivaloyl, mesitoyl), and per-silylated (tert-butyl-dimethyl silyl) glucose--glucose disaccharide derivatives has been reported.     

Susceptibility of captive adult winter-run Chinook salmon Oncorhynchus tshawytscha to waterborne exposures with infectious hematopoietic necrosis virus (IHNV)

Sexually mature female Chinook salmon Oncorhynchus tshawytscha with no prior history of exposure to infectious hematopoietic necrosis virus (IHNV) were susceptible to experimental infection induced by additions of virus to the water. The resulting infections resembled those observed among naturally infected hatchery and wild populations of Chinook salmon. Virus was detected as early as 4 d post-exposure (p.e.) and subsequently in all virus-exposed fish that died or that were examined at 14 d p.e. when the study was terminated. The greatest concentrations of virus, up to 10(8) plaque-forming units (pfu) ml(-1), were found in the ovarian fluid at 13 to 14 d p.e., but the virus was also found in high concentrations in the gill, kidney/spleen and plasma. In contrast, the virus was not recovered from unexposed control adult salmon that died or were sampled at the end of the study. Despite detecting concentrations of IHNV in excess of 10(7) pfu g(-1) of tissue, no specific microscopic lesions were found in IHNV-exposed compared to unexposed control salmon. The results of this initial study suggest that virus in the spawning environment, either from adult salmon or other sources, may contribute to its rapid spread among adult Chinook salmon, thereby considerably increasing the prevalence of IHNV infection in both wild and hatchery populations of adult Chinook salmon.     

Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues

Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, an intracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pg for purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stages of the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from the kidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over the course of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captive broodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from other tissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of the parasite.