Isolation and Characterization of Bile Acid 7-Dehydroxylating Bacteria from Human Feces

Methods for isolation of fecal 7α-dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7-dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C-7α and/or C-7β. All the isolates were strictly anaerobic, Gram-positive rods. Thirteen isolates were non-sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase-positive but indole-, lecithinase-, urease- and oxidase-negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7α-dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C-7α. Nine strains (10, 12, 36S, M-2, M-17, M-18, Y-98, Y-1112, and Y-1113) of the 7α-dehydroxylating bacteria were confirmed to have 7β-dehydroxylation ability, but five strains (O-51, O-52, O-71, O-72, and Y-67) could not transform ursodeoxycholic acid to lithocholic acid.     

Bile acid induction specificity of 7α-dehydroxylase activity in an intestinal Eubacterium species

The addition of cholic acid to growing cultures of $\text{Eubacterium}$ species V.P.I. 12708 caused a 25 and 46-fold increase in 7α-dehydroxylation activity using cell extracts or whole cell suspensions, respectively. Bile acid conversion rates using either [¹⁴C]-cholic acid or [¹⁴C]-chenodeoxycholic acid as substrates increased at approximately the same rate when either cholic or chenodeoxycholic acid was added to growing cultures as inducer. The induction of 7α-dehydroxylase activity was highly specific requiring a free C-24-carboxyl group and an unhindered 7α-hydroxy group on the B ring of the steroid nucleus. Unexpectedly, cholic acid also rapidly induced NADH:flavin oxidoreductase activity in growing cultures of this bacterium.     

Bile acid biosynthesis: the metabolism of 7 alpha-hydroxy-4-cholesten-3-one in the bile fistula rat.

The two primary bile acids, cholic acid (3α,7α,12α-tri-hydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholan-24-oic acid), are initially synthesized by way of identical precursors, and the point of divergence of this pathway is thought to occur at the intermediate 7α-hydroxy-4-cholesten-3-one. In order to test this hypothesis, bile fistula rats received simultaneous intra-venous infusions of ³H-7α-hydroxy-4-cholesten-3-one and ¹⁴C-cholesterol (5-cholesten-3β-ol). Assays of equal specific activities of the two bile acids from an infusion of ¹⁴C-cholesterol demonstrated the achievement of a steady state, and assays of equal specific activities from an infusion of ³H-7α-hydroxy-4-cholesten-3-one would-validate the above postulate. However, the infusion of ³H-7α-hydroxy-4-cholesten-3-one resulted in unequal specific activities in the bile acids of the rats investigated, with cholic acid always of a lower value. These results suggest that either 7α-hydroxy-4-cholesten-3-one is not the last common intermediate in the production of cholic acid and chenodeoxycholic acid, or that the infused bile acid intermediate was not metabolized in a fashion similar to that formed in the liver from cholesterol.     

Characteristics of bacteria isolated by the anaerobic roll-tube method from cheeses and ground beef.

In this study the methods of Hungate were used to quantitate the anaerobic bacteria present in commercially available ground beef, cheddar cheese, and German hand cheese. Of 235 anaerobic roll-tube isolates from ground beef and German hand cheese, all were facultative anaerobes. Of 213 anaerobic roll-tube isolates from cheddar cheese, 91% were facultative anaerobes and 9% were obligate anaerobes. Using results of biochemical tests, 14 or the 17 obligately anaerobic isolates from cheddar cheese were Propionibacterium acnes, two were strains of Propionibacterium that could not be speciated, and one was tentatively identified as a strain of Streptococcus evolutus. Obligate anaerobes were estimated to be present in the cheddar cheese at a level of about 10(6)/g. The possible significance of these levels of P. acnes in nonsterile foods is discussed.     

A quantitative evaluation of the conversion of 25-hydroxycholesterol to bile acids in man

The present study was directed toward providing additional information in man on the nature of a potential alternative pathway to cholic acid not involving an initial 7α-hydroxylation of cholesterol. Two bile fistula patients and one normal subject each received 25-hydroxy[G-³H]cholesterol; [¹⁴C]cholic and [¹⁴C] chenodeoxycholic acids were also simultaneously administered to one bile fistula patient and normal subject. The labeled 25-hydroxycholesterol was found to be poorly converted to primary bile acids by all three patients; the range of conversion was 9.7 to 18.9%. Cholic acid was favored over chenodeoxycholic acid by a margin of about 1.4/1. It is concluded that a pathway to primary bile acid via the 25-hydroxylation of cholesterol is of minor importance under conditions of normal or accelerated synthesis in man.     

Metabolism of 3α, 7α-dihydrexy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyi-5β-cholanoic acid in hamsters

The metabolic fate of the bile add analogs, 3α,7α-dihydroxy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, was investigated and compared with that of chenodeoxycholic acid in hamsters. Both bile acid analogs were absorbed rapidly from the intestine and excreted into bile at similar to that of chenodeoxycholic acid. In the strain of hamster studied, the biliary bile were conjugated with both glycine and taurine. After continuous intravenous infusion, chenodeoxycholic acid the analogs became the major bile acid constituents in bile. After oral administration of a single dose of these compounds, fecal analysis revealed the existence of unchanged material (25–35%) as well as considerable amounts of metabolites (65–75%). The major metabolites excreted into feces were more polar than the starting material and were tentatively identified as trifaydroxy-7-methyl compounds by radioactive thin-layer chromatography. However, monohydroxy compounds were also found in the fecal extracts. These results show that chenodeoxycholic acid and ursodeoxycholic acid with a methyl group at the 7-position are resistant to bacterial 7-dehydroxylation than the normally occurring bile acids and that a certain proportion of these analogs is hydroxylated to give the corespondiag trihydroxy compound(s), In a control experiment, about 5% of administered chenodeoxychoulic acid was metabolized to a trihydroxy feile acid, but most of the compound (95%) was transformed into lithocholic acid.     

Determination of mercury and organomercurial resistance in obligate anaerobic bacteria

A methodology for determining the minimum inhibitory concentration of inorganic and organomercurial compounds for obligate anaerobic bacteria is described. A wide variation in the susceptibility of anaerobic clinical and sewage isolates was observed. Isolates of Bacteroides ruminicola and Clostridium perfringens resistant to mercury were examined for their plasmid content and ability to demonstrate inducible resistance. None of the resistant anaerobes contained any plasmids, while resistant facultative isolates from the same source contained several plasmids. In 24 h, resistant strains of clostridia and Bacteroides volatilized 20 and 43% of the 203Hg2+ added to cultures, while Escherichia coli R100 and a sewage isolate of Enterobacter cloacae volatilized 63 and 27%, respectively, of the added 203Hg2+. Attempts to induce mercury resistance in the aerobic isolates were successful, but no induction was seen in the anaerobes. Thus, mercury resistance in these anaerobic isolates was neither inducible nor plasmid mediated.     

Propionate Enhances Synthesis and Secretion of Bile Acids in Primary Cultured Rat Hepatocytes via Succinyl CoA

To discover the role of propionate produced by colonic bacteria, this study examined the secretion of bile acids and cholesterol 7α-hydroxylase activity in the primary cultured hepatocytes. Addition of propionate (2 mM) to the medium for 48 h caused an increase in the bile acid secretion and enzyme activity, while acetate and butyrate had no significant influence. Bile acid secretion was increased by the addition of succinyl CoA and its precursor substances (α -ketoglutarate, valine, isoleucine, and methionine), but not malate and oxaloacetate, which are the metabolites of succinyl CoA. α -Ketoglutarate and valine also increased the activity of cholesterol 7α -hydroxylase. Since cholesterol 7α -hydroxylase is a microsomal cytochrome P-450 enzyme and the formation of δ-aminolevulinate from succinyl CoA in the mitochondria is the rate-controlling step for the subsequent synthesis of heme proteins, propionate may affect bile acid synthesis via elevation of mitochondrial succinyl CoA.     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.     

Isolation and characterization of a new osmotolerant, non-virulent Klebsiella pneumoniae strain SAP for biosynthesis of succinic acid

In the present study, isolation of anaerobic bacteria from 24 different eco-niches was carried out. A total number of 300 bacterial isolates, including 230 obligate and 70 facultative anaerobes were obtained using anaerobic techniques. All the isolates were initially screened for succinic acid production by Fluorescein test and TLC method. During screening, 10 isolates found to produce succinic acid were further examined by HPLC and then finally confirmed for succinic acid by LC-MS analysis. Amongst 10 isolates, isolate SAP, a facultative anaerobe isolated from buffalo rumen fluid, showed maximum yield of 2.1 g/l of succinic acid from 10 g of glucose in 24 hr under anaerobic condition. This isolate was identified as Klebsiella pneumoniae strain SAP by 16S rDNA sequence and signature sequence analysis. Mouse lethality test for the strain SAP showed LD50 value of 3.3 x 10(8) CFU/ml, which shows non-virulent nature of the strain. This strain may become a candidate strain for succinic acid production because of its osmotolerant nature and higher succinate:acetate ratio.     

Bile acid synthesis in cell culture

Confluent cultures of Hep G2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 +/- 8.6 micrograms/96 h, a rate more than 7-fold greater than that for cholic acid. Addition of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol but not the -3 alpha, 7 alpha-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12 alpha-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 micrograms/96 h for lithocholic and 3 alpha-hydroxy-5-cholenoic acids, respectively. Incubation of Hep G2 cells in medium containing 25% D2O permitted a comparison of the precursor/product relationship of cholesterol with 3 beta-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid.     

Effect of bile acids on formation of the mutagen, quercetin, from two flavonol glycoside precursors by human gut bacterial preparations

Human fecal cultures, induced with either of the flavonols, quercitrin or rutin, were grown in the presence of various concentrations of chenodeoxycholic acid, deoxycholic acid or cholic acid. Cell-free preparations (fecal preparations) from these cultures were then incubated with rutin or quercitrin. The formation of the aglycone, quercetin, was monitored by the Ames assay using tester strain TA98. The presence of chenodeoxycholic or deoxycholic acids in the quercitrin-induced culture resulted in a fecal preparation which enhanced the mutagenesis of quercitrin approximately two-fold at optimal concentrations of 0.6 mM and 0.8 mM respectively. Higher concentrations of these bile acids decreased the activity of the fecal preparations. Cholic acid gave similar results except a much higher concentration (3.0 mM) was required to achieve this effect. Analogous results with rutin-induced cultures were less clear cut: considerable variation in bile acid effect was noted among volunteers. The authors propose that bile acid in the medium may enhance the ability of rutin- and quercitrin-glycosidase elaborating organisms to successfully compete with other microbial populations. Additionally, the greater variation in results using rutin as inducer may reflect more heterogeneous populations of organisms active against this substrate. The possible role of bile acids and flavonols in bowel cancer is discussed.     

A rapid non-chromatographic analysis of individual bile acids in human bile extracts

It is postulated that the six conjugated bile acids of most common occurrence in human bile could be analyzed by three enzymic and one chemical assay without any prior chromatographic separation of the bile acids. In health, all bile acids in liver or gall bladder bile are conjugated with either glycine or taurine and have an a-hydroxyl group at the 3 position. In addition, the trihydroxy bile acid, cholic (C) has a 7α- and a 12α-hydroxy group while the dihydroxy bile acids either have a second hydroxyl group at the 7α-position (chenodeoxycholic acid, CDC) or at the 12α-position (deoxycholic acid, DC). Hydroxysteroid dehydrogenases (HSDH) specific for oxido-reductase activity at the 3α-, 7α- and 12α-positions would directly quantify these 3α-, 7α- and 12α-hydroxyl groups in a sample of bile or bile extract. Subsequent data would be used to solve three simultaneous equations yielding solutions for the overall concentrations of conjugated C, conjugated CDC and conjugated DC on the assumption that the overall concentration of lithocholic acid is negligible (< 2 %). A suitable assay for the sulphonate group containing taurine conjugates, such as that described by Christie, Macdonald & Williams, 1975, along with the total bile acid measurement would readily facilitate the estimation of the glycine/taurine ($\text{G}\text{T}$) ratio. This ratio applied to the enzymatically derived estimates for conjugated DC, CDC and C would approximate the glycodeoxycholate (GDC), glycochenodeoxycholate (GCDC), glycocholate (GC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC) and taurocholate (TC) concentrations. Figures for these concentrations would be based on the assumption that the $\text{G}\text{T}$ ratio is approximately the same for each bile acid and that all the bile acids are conjugated.     

Inhibition of Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> by Continuous-Flow Cultures of Human Stool Microflora With and Without Anaerobic Gas Supplementation

A continuous-flow competitive exclusion (CFCE) culture model of human stool microflora was used to examine whether supplemental anaerobic gas is necessary for maintenance of anaerobes and inhibition of vancomycin-resistant Enterococcus (VRE). CFCE cultures of human stool microflora were maintained with supplemental nitrogen, without supplemental nitrogen, or with percolated room air. Cultures with or without supplemental nitrogen maintained >9 log₁₀ CFU mL^–1 of obligate anaerobes and eliminated 10⁶ CFU mL^–1 of VRE. When room air was percolated into the culture, anaerobes were detected at 2 log₁₀ CFU mL^–1, and the same VRE inoculum was not eliminated (P < 0.001). These data demonstrate that human stool CFCE cultures maintain high levels of obligate anaerobes and inhibit VRE without the addition of supplemental anaerobic gas.     

Effect of bile acid oxazoline derivatives on microorganisms participating in 7 alpha-hydroxyl epimerization of primary bile acids

We tested bile acid oxazoline derivatives of chenodeoxycholic (CDC-OX), 7-ketolithocholic (7-KLC-OX), ursodeoxycholic (UDC-OX), and deoxycholic (DC-OX) as inhibitors of the 7-epimerization of the primary bile acids cholic acid (CA) and CDC in cultures of four species of bacteria and the human fecal flora. The organisms tested elaborate a 7 alpha- and/or 7 beta-hydroxysteroid dehydrogenase (HSDH); they were Escherichia coli (7 alpha-HSDH), Bacteroides fragilis (7 alpha-HSDH), Clostridium absonum (7 alpha- and 7 beta-HSDH) and Eubacterium aerofaciens (7 beta-HSDH). None of the oxazolines affected 7 alpha-OH oxidation of CA or CDC by E. coli or the growth of the organism. All the oxazolines (except UDC-OX) inhibited the growth of B. fragilis and its 7 alpha-HSDH. In contrast, only DC-OX blocked 7 alpha-OH epimerization of CA by C. absonum. Surprisingly, the other three oxazolines enhanced 7 alpha-OH epimerization of CA, but not that of CDC, which was inhibited (CDC-OX greater than 7-KLC-OX much greater than UDC-OX). Enzymic data suggest that CDC-OX in the presence of CA can induce a greater level of both 7 alpha- and 7 beta-HSDH than CA or CDC-OX alone, CDC-OX being more toxic in the presence of CDC. Formation of urso-bile acid from 7-keto substrates by E. aerofaciens is totally blocked by the oxazolines (except UDC-OX). Similarly, suppression of urso-bile acid formation from primary bile acids by the human fecal flora was evident with DC-OX greater than 7-KLC-OX greater than CDC-OX much greater than UDC-OX, the last being ineffective. The inhibitory activity of the oxazolines on the 7-dehydroxylation of primary bile acids by human fecal flora followed the same order.     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

Chenodeoxycholic acid synthesis in the hamster: A metabolic pathway via 3β, 7α-dihydroxy-5-cholen-24-oic acid

The quantitative significance of the metabolism of 3β, 7α-dihydroxy-5-cholen-24-oic acid to chenodeoxycholic acid was evaluated in the hamster. A precursor-product relationship was established in this species by the finding that intravenous administration to an animal previously given cholesterol-4-¹⁴C caused a significant reduction in the specific activity of chenodeoxycholic acid. Administration of 12.9 μmole of the precursor was followed by a 10-fold increase in chenodeoxycholic acid excretion although the predominant excretory pathway was via biliary excretion as a monosulfate. The data indicate that synthesis of bile acid from cholesterol via the intermediate 3β, 7α-dihydroxy-5-cholen-24-oic acid can be a quantitatively important pathway.     

Characterization of some anaerobic bacteria from the liquid phase of a mesophilic anaerobic digester fed with a prefermented cheese whey substrate

Bacterial counts on the contents of an anaerobic fixed-bed digester receiving a whey substrate were conducted using the modified roll tube technique. Average anaerobic counts after 48 hours incubation on lactate containing media were 3.12 × 10⁹ and 3.7 × 10⁹ ml^–1, respectively. These counts were between 140 and 190 times higher than aerobic counts on the same media. Seventy-four strains from both media were isolated and characterized, and the relationship between the organisms was calculated according to the similarity coefficient of Sokal and Michener [20]. The organisms were clustered using the unweighted pair group method and the results were presented in the form of a simplified dendrogram. The isolates clustered in three major groups (A, B, and C) at a similarity level of 76%. A small diffuse group of five organisms was also found. The organisms in two (A and B) of the major clusters were obligate anaerobes. Cluster A represented 34% of the isolates, cluster B represented 50%, and the facultative streptococci in cluster C, 11%. The isolates in clusters A and B could only tentatively be identified as members of the generaBacteroides andPeptostreptococcus, respectively. The isolates could not be positively identified.