Biomedical subjects

C E Cerniglia

Publications and source records attributed to C E Cerniglia.

At least 19 recordsLinked to original sources

PubMed2006

Echinacea purpurea supplementation stimulates select groups of human gastrointestinal tract microbiota.

BACKGROUND AND OBJECTIVE: The objective of this research was to determine the effects of the dietary supplement Echinacea purpurea on aerobic and anaerobic bacteria common to the human gastrointestinal (GI) tract. Botanical extracts have shown in vitro antimicrobial effects against certain pathogenic bacteria. It is uncertain if medicinal herbs have any effect against pathogenic bacteria or on the native GI microbiota. METHODS: Fifteen human subjects consumed 1000 mg of standardized E. purpurea for 10 days. Faecal samples were collected at baseline, 10 days and 17-18 days following supplementation. Samples were tested for select aerobic and anaerobic bacteria using plate culture microbiological methods. RESULTS AND DISCUSSION: Significant increases were found for total aerobic bacteria, Bacteroides group and Bacteroides fragilis after E. purpurea exposure. Supplementation did not significantly alter the number of enteric bacteria, enterococci, lactobacilli, bifidobacteria or total anaerobic bacteria. CONCLUSION: Echinacea supplementation has altered the GI microbiota. The health consequences associated with this change are unknown but previous research has shown increased Bacteroides concentrations associated with diarrhoea, inflammatory bowel disease and increased risk of colon cancer. Additional research should delineate the role of Echinacea in the stimulation of Bacteroides and describe the effects of other botanical supplements to the GI microbiota.

Adult↗

PubMed2005

Approaches in the safety evaluations of veterinary antimicrobial agents in food to determine the effects on the human intestinal microflora.

The administration of antimicrobial agents to livestock creates potential for antibiotic residues to enter the food supply and be consumed by humans. Therefore, as a process of food animal drug registration, national regulatory agencies and international committees evaluate data regarding the chemical, microbiologic, pharmacokinetic, pharmacodynamic, pharmacologic, toxicologic, and antimicrobial properties of veterinary drugs to assess the safety of ingested antimicrobial residues to consumers. Currently, European, Australian and United States guidelines for veterinary drug registration require a safety assessment of microbiologic hazards from consumption of antimicrobial residues taking into account the potentially adverse effects on human intestinal microflora. The main concerns addressed are selection of resistant bacteria in the gastrointestinal tract and disruption of the colonization barrier of the resident intestinal microflora. Current requirements differ among national agencies. Efforts are ongoing internationally to review and harmonize approaches and test methods and protocols for application to these microbiologic safety evaluations of antimicrobial drug residues in food. This review describes the background to current regulatory approaches used in applying in vitro and in vivo methods to set a microbiologic acceptable daily intake for residues in food derived from animals treated with an antimicrobial agent. This paper also examines the current research needs to support these evaluations.

Animals↗

PubMed2004

Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources.

This study evaluated the molecular diversity of 29 Salmonella serotypes isolated from turkey ceca and the production environment. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), sulfamethoxazole/trimethoprim (SXT) (3%) and tobramycin (3%). The minimum inhibitory concentration (MIC) values ranged from 32 to >/=1024 microg/ml. The pulsed-field gel electrophoresis (PFGE) and ribotyping patterns were identical within each of the serotypes Heidelberg, Worthington and Muenster. The plasmid profiles were identical within each of the Salmonella serotypes. Two different clones of Salmonella anatum were differentiated by PFGE typing but not by ribotyping. Heidelberg isolates from nine turkey ceca and three drinker samples had identical antibiotic resistance, PFGE, ribotype and plasmid patterns, suggesting that transmission of this particular clone may have occurred between the birds and the drinkers. Identical PFGE, ribotype and plasmid patterns were observed in one Salmonella worthington isolate from turkey ceca in one flock and two S. worthington isolates from feeder contents and drinkers from a subsequent flock, suggesting transmission of this pathogen between flocks. Individual and multiple polymerase chain reaction (PCR) analyses revealed the presence of the virulence genes invA, aceK and sopB and the absence of the h-1i gene in all isolates. A combination of genotypic and phenotypic markers can be useful in studying genetic variation among natural salmonellae populations in turkey production and delineating possible transmission pathways.

Animal Husbandry↗

PubMed2003

Phenotypic and genotypic characterization of competitive exclusion products for use in poultry.

AIMS: Phenotypic and genotypic bacteria identification methods were compared for their efficacy in determining the composition of competitive exclusion (CE) products. METHODS AND RESULTS: Phenotypic methods used for bacterial identification were fatty acid methyl ester profiles, biochemical assays and carbohydrate utilization profiles. Genotypic methods were MicroSeq16S rRNA sequence analysis and BLAST searches of the GenBank sequence database. Agreement between phenotypic and genotypic methods for identification of bacteria isolated from the Preempt CE product was 20%. A defined test mixture of bacteria was identified to the species level 100% by BLAST analysis, 64% by MicroSeq and 36% by phenotypic techniques. CONCLUSIONS: The wide range of facultative and obligate anaerobic bacteria present in a CE product are more accurately identified with 16S rRNA sequence analyses than with phenotypic identification techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will provide guidelines for manufacturers of CE products to submit more reliable product information for market approval by regulatory agencies.

Animals↗

PubMed2003

Degradation of phenanthrene and naphthalene by a Burkholderia species strain.

Burkholderia sp. TNFYE-5 was isolated from soil for the ability to grow on phenanthrene as sole carbon and energy source. Unlike most other phenanthrene-degrading bacteria, TNFYE-5 was unable to grow on naphthalene. Growth substrate range experiments coupled with the ring-cleavage enzyme assay data suggest that TNFYE-5 initially metabolizes phenanthrene to 1-hydroxy-2-naphthoate with subsequent degradation through the phthalate and protocatechuate and beta-ketoadipate pathway. A metabolite in the degradation of naphthalene by TNFYE-5 was isolated by high-pressure liquid chromatography (HPLC) and was identified as salicylate by UV-visible spectral and gas chromatography-mass spectrometry analyses. Thus, the inability to degrade salicylate is apparently one major reason for the incapability of TNFYE-5 to grow on naphthalene.

Biodegradation, Environmental↗

PubMed2002

Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples.

A membrane-array method was developed for the detection of human intestinal bacteria in fecal samples without using the expensive microarray-arrayer and laser-scanner. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to nitrocellulose membranes. Digoxigenin (DIG)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or pure cultured bacteria using two universal primers, and were hybridized to the membrane-array. Hybridization signals were read by NBT/BCIP color development. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, B. vulgatus, B. fragilis, B. distasonis, Clostridium clostridiiforme, C. leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, R. bromii, R. callidus, R. albus, Bifidobacterium longum, B. adolescentis, B. infantis, Eubacterium biforme, E. aerofaciens, Lactobacillus acidophilus,Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the membrane-array method is a reliable technique for the detection of predominant human intestinal bacteria in the fecal samples. The result was also confirmed by using specific PCR methods for these bacteria.

Adult↗

PubMed2001

Degradation of biphenyl by Mycobacterium sp. strain PYR-1.

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.

Benzoic Acid↗

PubMed2001

Metabolism of anthracene by a Rhodococcus species.

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.

Anthracenes↗

PubMed2001

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans.

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.

Amino Acid Sequence↗

PubMed2001

DNA packaging and developmental intermediates of a broad host range Vibrio vulnificus bacteriophage 71A-6.

The structural intermediates in the capsid assembly and DNA packaging pathway of Vibrio vulnificus bacteriophage 71A-6, a rod-shaped double-stranded DNA podovirus, were isolated by ultracentrifugation and studied by electron microscopy, SDS-PAGE and pulsed-field gel electrophoretic analysis. Bacteriophage 71A-6 synthesized rod-shaped capsids (mean length=200+/-8 nm; mean width=47+/-3 nm n=50) during its development. Several host proteins that probably help in the assembly and maturation of the capsids were attached to these capsids as spherical structures. A capsid-DNA or DNA packaging complex that consisted of the mature capsids, DNA and a 42.5-kDa protein was also isolated. The size of the capsids increased in length and decreased in width (mean length=220+/-8 nm; mean width=45+/-3 nm n=50) either during or after the DNA packaging. The capsid fractions contained about 12 phage structural proteins and eight host proteins. At least three proteins were tentatively identified: a 38.5-kDa major capsid protein, a 35.2-kDa tail protein and 42.5-kDa packaging initiator or terminator protein. The size of the bacteriophage 71A-6 genome was determined to be 143.0-kb by pulsed-field gel electrophoresis. The total mass of all the mature phage proteins corresponded to only 14.0% of the coding capacity of phage genome.

Bacteriophages↗

PubMed2001

Identification of predominant human and animal anaerobic intestinal bacterial species by terminal restriction fragment patterns (TRFPs): a rapid, PCR-based method.

Identification of predominant human and animal intestinal tract anaerobes by conventional methods is cumbersome, time-consuming and less sensitive as compared to molecular methods. We have developed a molecular technique to identify most of the abundant intestinal microflora by polyermase chain reaction (PCR) amplification of a 16S rRNA gene fragment using a pair of universal PCR primers. The forward PCR primer was labelled with 6-carboxyfluorescein amino hexy (6-FAM) fluorescent dye to detect the terminal fragment of the PCR products after digestion with restriction enzymes. The PCR products were purified and digested with restriction enzymes and were analysed by capillary electrophoresis using an automated DNA sequencer. The data was analysed with GeneScan software 2.1. Eleven bacteria (Eubacterium biforme, E. limosum, Peptostreptococcus productus, Lactobacillus acidophilus, Bacteroides thetaiotaomicron, B. vulgatus, B. distasonis, Clostridium clostridiiforme, C. leptum, C. perfringens and Escherichia coli) that are predominant in human and animal intestinal tract were successfully identified by this rapid molecular technique. This protocol is rapid, accurate, sensitive and capable of identifying multiple organisms in a single sample.

Animals↗

PubMed2001

A rapid method for determining the tuberculocidal activity of liquid chemical germicides.

A rapid, quantitative method has been developed for determining the tuberculocidal activity of liquid chemical germicides. In this method, a test strain of Mycobacterium bovis that carries the firefly luciferase gene is exposed to a germicide, and the surviving bacteria are detected by bioluminescence. The tuberculocidal activities of five commercially available glutaraldehyde-based disinfectants were tested, and all reduced the number of surviving mycobacteria by greater than five orders of magnitude. In contrast, a phenol-based disinfectant with tuberculocidal claims gave less than one order of magnitude reduction of the test organism. With this method for determining tuberculocidal activity, results can be obtained in less than one day, compared with weeks or months for the standard tuberculocidal assays.

Bacteriological Techniques↗

PubMed2001

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1.

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.

Anthracenes↗

PubMed2001

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1.

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.

Cloning, Molecular↗

PubMed2001

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides.

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.

Biodegradation, Environmental↗

PubMed2001

Biotransformation of malachite green by the fungus Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.

Biodegradation, Environmental↗

PubMed2001

Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs.

This study validated a polymerase chain reaction-based method for the detection of a specific bovine mitochondrial gene derived from rendered bovine tissues and admixed with complete animal feed. Four laboratories participated in this effort: one state laboratory and three Food and Drug Administration (FDA) laboratories, including one FDA field laboratory. The protocol used a statistical approach of 90% probability, with a 95% confidence interval for determining acceptable rates of false-positive and false-negative samples. Each participating laboratory analyzed 30 samples of feed each containing 0, 0.125, and 2.0% bovine meat and bone meal (BMBM), for a total of 90 feed samples. The samples were randomized such that the analysts were unaware of the true identity of the test samples. The results demonstrated that all laboratories met the acceptance criteria established for this protocol. The overall rates of false-negative results were 0.83% (1/120) at the level of 0.125% BMBM and 1.67% (2/120) at the level of 2% BMBM. The overall rate of false-negative results for all levels of BMBM was 1.25% (3/240). The rate for false-positive results was 0.83%.

Animal Feed↗

PubMed2000

Cloning, sequencing, and expression in Escherichia coli of a cytochrome P450 gene from Cunninghamella elegans.

A polyclonal antibody against microsomes of a fungus, Cunninghamella elegans, was used to screen a C. elegans cDNA library. A cDNA clone, containing an open reading frame (ORF) encoding a protein of 389 amino acids (aa), was obtained. GenBank comparison (BLAST) showed that the protein was closely related to P450 because a heme-binding region, which is highly conserved in all P450 sequences, was found in the ORF protein. Using an oligo probe designed from this C. elegans heme-binding region to rescreen the cDNA library, we obtained three new clones. Sequence comparison showed that the three clones, with different length cDNA inserts, were from the same mRNA of the C. elegans P450 gene. One clone had the full C. elegans P450 gene, encoding 473 aa with a molecular mass of 54958.60, whereas the 389 was a part of the 473 aa without the N-terminal. The entire C. elegans P450 gene was successfully subcloned and overexpressed in a plasmid-Escherichia coli system (pQE30). Immunostaining with three antibodies (CYP1A1, CYP2E1, and CYP3A1) against mammalian P450 enzymes and benzidine staining for hemoproteins showed positive results for the recombinant protein expressed in E. coli. A phylogenetic tree was constructed by comparison of other fungal P450s to the C. elegans sequence. The C. elegans P450 clustered close to the cyp51 family and was named cyp509A1 by the International Committee on the Nomenclature for Cytochrome P450 Enzymes.

Amino Acid Sequence↗