Fatemeh Rafii

Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identification of whole bacteria, either by comparison with archived reference spectra or by co-analysis with cultures of known bacteria. Bacteria were sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In the first experiment, both bacterial strains that had been previously analyzed to obtain reference spectra and other strains that had not been analyzed were blind-numbered and their spectra were obtained. Those strains that matched reference spectra were found to be correctly identified. A second experiment involved co-analysis of reference strains and bind-numbered strains under identical conditions; species-specific identification was demonstrated by comparison of spectra of the blind-numbered strains with those of the standards. In all of the spectra obtained in these experiments, each bacterial strain showed a few characteristic high-mass ions which are thought to be derived from bacterial proteins. This work represents the first reported instance of successful bacterial chemotaxonomy by MALDI-TOFMS analysis of whole cells. For the strains tested, the method is rapid and simple.

Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin.

Abstract. Fecal bacteria from a healthy individual were screened for the specific bacteria involved in the metabolism of dietary isoflavonoids. Two strains of bacteria capable of producing primary and secondary metabolites from the natural isoflavone glycosides daidzin and genistin were detected. The metabolites were identified by comparison of their HPLC/mass, 1H NMR and UV spectra with those of standard and synthetic compounds. Both Escherichia coli HGH21 and the gram-positive strain HGH6 converted daidzin and genistin to the their respective aglycones daidzein and genistein. Under anoxic conditions, strain HGH6 further metabolized the isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein, respectively. The reduction of a double bond between C-2 and C-3 to a single bond was isoflavonoid-specific by strain HGH6, which did not reduce a similar bond in the flavonoids apigenin and chrysin. Strain HGH6 did not further metabolize dihydrodaidzein and dihydrogenistein. This is the first study in which specific colonic bacteria that are involved in the metabolism of daidzin and genistin have been detected.

Variations in metabolism of the soy isoflavonoid daidzein by human intestinal microfloras from different individuals

Isoflavonoids found in legumes, such as soybeans, are converted by intestinal bacteria to metabolites that might have increased or decreased estrogenic activity. Variation in the effects of dietary isoflavonoids among individuals has been attributed to differences in their metabolism by intestinal bacteria. To investigate this variation, the metabolism of the isoflavonoid daidzein by bacteria from ten fecal samples, provided at different times by six individuals on soy-containing diets, was compared. After anaerobic incubation of bacteria with daidzein for 2 weeks, four samples had metabolized daidzein and six samples had not. Three of the positive samples were from individuals whose microflora had not metabolized daidzein in previous samples. Dihydrodaidzein was observed in one sample, dihydrodaidzein and equol in another sample, and equol and O-desmethylangolensin in two other samples. These results corroborate the hypothesis that the microflora of the gastrointestinal tract of an individual influences the particular isoflavone metabolites produced following consumption.

COMPARISON OF ESSENTIAL OILS FROM THREE PLANTS FOR ENHANCEMENT OF ANTIMICROBIAL ACTIVITY OF NITROFURANTOIN AGAINST ENTEROBACTERIA

Background: Piperitone from plant essential oils enhancesbactericidal activities of nitrofurantoin and furazolidone against bacteria from the family Enterobacteriaceae. In this study, the essential oils of spearmint (Mentha spicata L.), dill (Anethum graveolens L.) and peppermint (Mentha piperita L.)were screened for augmentation of nitrofurantoin activity and the most active components were determined. Method: The effects of essential oils and their components on the bactericidal activity of nitrofurantoin against Enterobacter cloacae were studied using disk-diffusion and agar-dilution methods. The composition of essential oils was studied using gas chromatography-mass spectrometry. Results:M. spicata and A. graveolens oils exhibited the highest effects. Gas chromatography-mass spectrometry analysis showed that the oils of these two plants contained 40.12 and 20.32% carvone, respectively. Pure carvone and piperitone equally increased the bactericidal activity of nitrofurantoin. Other ingredients of essential oils, including camphor, limonene and menthone, were less effective.

Cloning and expression in Escherichia coli of an azoreductase gene from Clostridium perfringens and comparison with azoreductase genes from other bacteria.

A genomic library of Clostridium perfringens ATCC 3626 was constructed in phage λgt11 and screened with an antibody against the C. perfringens azoreductase, which catalyzes the reduction of azo dyes to aromatic amines. A positive recombinant phage, containing a 3.8 kb DNA fragment insert was selected and purified. Lytic and lysogenic Escherichia coli cultures infected with the recombinant phage had higher azoreductase activity than cultures infected only with the vector λgt11. The 3.8 kb DNA fragment was amplified by PCR and found to hybridize with one band from C. perfringens DNA digested with EcoR1, indicating the presence of a single copy of the azoreductase gene. The fragment also hybridized with DNA from other azoreductase‐producing Clostridium species, a Eubacterium sp., Enterobacter cloacae, Citrobacter amalonaticus and E. coli, but not with DNA from some other species of anaerobic bacteria capable of reducing azo dyes. The data indicate that the sequence of the azoreductase gene of C. perfringens is conserved in some anaerobes and facultative anaerobes, but not in others, and that different types of azoreductase genes must be found in other anaerobic bacteria.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric detection of bacterial biomarker proteins isolated from contaminated water, lettuce and cotton cloth.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of bacterial proteins were obtained from water, lettuce and cloth samples contaminated with Shigella flexneri, Escherichia coli, and Aeromonas hydrophila. Spectra were obtained using proteins directly isolated from water (or water used for rinsing samples) without culturing the bacteria. For S. flexneri and E. coli, two marker ions for specific proteins associated with a virulence-related property (acid resistance) were easily detected. For A. hydrophila, ions from two specifically selected marker proteins, as well as ions from the larger group of proteins isolated from pure cultures, all matched spectra from a contaminated water sample, providing strong evidence that A. hydrophila was the bacterial contaminant. Rinse water from contaminated lettuce and cloth samples showed the same marker ions as the contaminated water samples.

Alterations in DNA Gyrase and Topoisomerase IV in Resistant Mutants of Clostridium perfringens Found after In Vitro Treatment with Fluoroquinolones

ABSTRACT To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants.

Mechanism of metronidazole-resistance by isolates of nitroreductase-producing Enterococcus gallinarum and Enterococcus casseliflavus from the human intestinal tract

Enterococcus casseliflavus and Enterococcus gallinarum strains resistant to metronidazole, nitrofurantoin and nitrofurazone were isolated from fecal samples of a patient with recurrent ulcerative colitis treated with metronidazole. Unlike other metronidazole-resistant bacteria, these strains produced nitroreductase but metabolized metronidazole to compounds that could not be detected by liquid chromatography with UV or mass spectral analysis. Metronidazole-susceptible Clostridium perfringens grew equally well in spent cultures of Enterococcus spp. incubated with or without metronidazole. These data indicate that the nitroreductases produced by these Enterococcus strains did not activate metronidazole to bactericidal metabolites and these bacteria may reduce the effectiveness of metronidazole. We have indirect evidence for an alternative pathway that results in metronidazole resistance. These strains of enterococcus had nitroreductase so resistance should not have occurred.

Survival of Shigella flexneri on vegetables and detection by polymerase chain reaction

Commercially prepared and packaged fresh vegetables were tested to determine the types and levels of indigenous microflora. Sixteen species of bacteria from 11 genera were identified and titers of up to 1 × 1010 cells per gram of vegetable were observed. To evaluate the survival of Shigella spp. on packaged vegetables, an avirulent insertion mutant of Shigella flexneri 5 (pHS 1059) was added to vegetables. This strain survived in phosphate-buffered saline at pH 7.3 at 5 to 10°C for more than 3 months. It also survived for several days at both ambient and refrigerator temperatures when inoculated onto various commercially prepared vegetables. A rapid method for detecting Shigella spp. on vegetables was developed by using the polymerase chain reaction (PCR) to amplify a 118-base-pair DNA fragment from the S. flexneri virulence-associated spa region. The PCR also generated the corresponding fragments from S. sonnei , S. boydii , and Shigella sp. This fragment was also observed when S. flexneri cells were used to artificially contaminate sterile and nonsterile vegetables, but no amplified fragment was observed when the normal microflora of the vegetables were eluted and tested by PCR.

The antimicrobial effects of selenium nanoparticle-enriched probiotics and their fermented broth against Candida albicans.

BackgroundLactic acid bacteria are considered important probiotics for prevention of some infections. The aim of this work was to investigate the effect of selenium dioxide on the antifungal activity of Lactobacillus plantarum and L. johnsonii against Candida albicans.MethodsLactobacillus plantarum and L. johnsonii cells, grown in the presence and absence of selenium dioxide, and their cell-free spent culture media were tested for antifungal activity against C. albicans ATCC 14053 by a hole-plate diffusion method and a time-kill assay.ResultsBoth L. plantarum and L. johnsonii reduced selenium dioxide to cell-associated elemental selenium nanoparticles. The cell-free spent culture media, from both Lactobacillus species that had been grown with selenium dioxide for 48 h, showed enhanced antifungal activity against C. albicans. Enhanced antifungal activity of cell biomass against C. albicans was also observed in cultures grown with selenium dioxide.ConclusionsSelenium dioxide-treated Lactobacillus spp. or their cell-free spent broth inhibited the growth of C. albicans and should be investigated for possible use in anti-Candida probiotic formulations in future.