Contribution of gut bacteria to the metabolism of cyanidin 3-glucoside in human microbiota-associated rats.

Cyanidin 3-glucoside (C3G) is one of the major dietary anthocyanins implicated in the prevention of chronic diseases. To evaluate the impact of human intestinal bacteria on the fate of C3G in the host, we studied the metabolism of C3G in human microbiota-associated (HMA) rats in comparison with germ-free (GF) rats. Urine and faeces of the rats were analysed for C3G and its metabolites within 48 h after the application of 92 µmol C3G/kg body weight. In addition, we tested the microbial C3G conversion in vitro by incubating C3G with human faecal slurries and selected human gut bacteria. The HMA rats excreted with faeces a three times higher percentage of unconjugated C3G products and a two times higher percentage of conjugated C3G products than the GF rats. These differences were mainly due to the increased excretion of 3,4-dihydroxybenzoic acid, 2,4,6-trihydroxybenzaldehyde and 2,4,6-trihydroxybenzoic acid. Only the urine of HMA rats contained peonidin and 3-hydroxycinnamic acid and the percentage of conjugated C3G products in the urine was decreased compared with the GF rats. Overall, the presence of intestinal microbiota resulted in a 3·7% recovery of the C3G dose in HMA rats compared with 1·7% in GF rats. Human intestinal bacteria rapidly degraded C3G in vitro. Most of the C3G products were also found in the absence of bacteria, but at considerably lower levels. The higher concentrations of phenolic acids observed in the presence of intestinal bacteria may contribute to the proposed beneficial health effects of C3G.

Impact of gut microbiota on intestinal and hepatic levels of phase 2 xenobiotic-metabolizing enzymes in the rat.

Using immunoblotting, we compared levels of phase 2 enzymes in liver, small intestine, cecum, and colon of germ-free and control rats (reassociated with rat intestinal microbiota). In addition, colonic levels were studied after association with human intestinal microbiota. The glutathione transferases (GSTs) studied, gastrointestinal glutathione peroxidase (GPX2), both epoxide hydrolases (EPHXs), and N-acetyltransferase (NAT) 1, were detected in all tissues. GPX2 and GSTP1 were highest in large bowel; the other enzymes of this group were highest in liver. NAT2 was found in the large bowel but not in the liver or small bowel. Sulfotransferases (SULTs) were detected in liver but were absent in small intestine; two forms were present at moderate levels in the large intestine. Strong gender-dependent differences were observed for several enzymes in liver but not in gut. Colonic levels in germ-free animals differed from those in control animals (* indicates statistical significance) for GSTA1/2 (4.0*- and 5.0*-fold in males and females, respectively), GSTA4 (1.5*/1.9*-fold), GSTM1 (1.1/1.5*-fold), EPHX1 (3.5*/2.4*-fold), EPHX2 (1.4/2.1*-fold), SULT1B1 (0.4*/0.6*-fold), SULT1C2 (1.3/1.6*-fold), and NAT2 (1.4/1.5*-fold). Smaller effects were observed when rats were colonized with human, compared with rat, intestinal bacteria. Cecal enzyme levels in germ-free rats were changed similarly to those in colon. No effects were seen in small intestine. In liver, SULT1A1, SULT1C1, and SULT1C2 were elevated in germ-free animals of both genders (1.5- to 2.6-fold); hepatic EPHX2 was elevated 1.6-fold in females. In conclusion, intestinal microbiota can affect levels of xenobiotic-metabolizing enzymes in large intestine and liver, but the effects observed were moderate compared with tissue-dependent expression differences.

The bioavailability of apigenin-7-glucoside is influenced by human intestinal microbiota in rats.

We investigated the impact of human intestinal microbiota on bioavailability of the flavone apigenin-7-glucoside (A7G) by comparing germ-free and human microbiota-associated (HMA) rats. First, the ability of the human intestinal microbiota to convert A7G was proven in vitro by incubating A7G with fecal suspensions. Apigenin, naringenin, and 3-(4-hydroxyphenyl)propionic acid were formed as main metabolites. After application of A7G to germ-free rats, apigenin, luteolin, and their conjugates were detected in urine and feces. In HMA rats, naringenin, eriodictyol, phloretin, 3-(3,4-dihydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propionic acid, and 4-hydroxycinnamic acid in their free and conjugated forms were additionally formed. In whole-blood samples from germ-free and HMA rats, only apigenin conjugates and phloretin, respectively, were detected. The total excretion of A7G and its metabolites within 48 h was similarly low in both germ-free and HMA rats, with 11 and 13% of the A7G dose, respectively. In germ-free rats, A7G metabolites dominated by apigenin and its conjugates were mainly excreted with feces. In contrast, the compounds in HMA rats were predominantly recovered from urine, 3-(4-hydroxyphenyl)propionic acid being the main metabolite. The ability of selected gut bacteria and the host intestinal mucosa to deglycosylate A7G was tested using cell extracts. Apigenin was formed by cytosolic extracts of Eubacterium ramulus and Bacteroides distasonis and by the microsomal fraction of the small intestinal mucosa of rats. Overall, human intestinal microbiota largely contributed to A7G metabolism, indicating its influence on the bioactivity of flavones.

Antibiotics in dust originating from a pig-fattening farm: a new source of health hazard for farmers?

Pig-house dust originates from feed, bedding, feces, and the animals themselves. If the animals receive drugs such as antibiotics, residues of these substances may occur in manure, in the air, or on surfaces of the respective animal house. In a retrospective study, we investigated dust samples collected during two decades from the same piggery for the occurrence of various antibiotics. In 90% of these samples, we detected up to five different antibiotics, including tylosin, various tetracyclines, sulfamethazine, and chloramphenicol, in total amounts up to 12.5 mg/kg dust. High dust exposure in animal confinement buildings is believed to be a respiratory health hazard because of the high content of microorganisms, endotoxins, and allergens. Further risks may arise from the inhalation of dust contaminated with a cocktail of antibiotics. Apart from that, our data provide first evidence for a new route of entry for veterinary drugs in the environment.

Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry.

Tetracyclines are widely used in farm animals. This can cause drug residues in products of animal origin and, after excretion of these substances, in animal slurry and in soil fertilized with that slurry. In this paper, we present a method based on a microbiological assay coupled with HPLC for the detection of oxytetracycline, tetracycline, and chlortetracycline in eggs. After a simple liquid extraction of the samples and HPLC separation, fractions were collected on microtiter plates, and the tetracyclines were analyzed using the Staphylococcus aureus assay. This method was able to identify residues of tetracyclines in eggs at a level set by regulatory agencies (i.e., 200 microg/kg). In addition, it was shown that the described microbiological method can be used as a screening assay for the detection of tetracyclines and possible biologically active metabolites in animal slurry and soil samples. Employing the same extraction procedure, it was demonstrated that LC-MS-MS allowed the quantification of 20-400 microg/kg in eggs with recoveries ranging from 71 to 109% and RSDs of 3-15%.

Recovery and metabolism of xanthohumol in germ-free and human microbiota-associated rats.

The impact of human intestinal bacteria on the bioavailability of the prenylflavonoid xanthohumol (XN) was studied by comparing germ-free (GF) and human microbiota-associated (HMA) rats. After XN application, XN, XN conjugates, and isoxanthohumol (IX) conjugates occurred in blood samples of GF and HMA rats, whereas IX was detected only in the blood of HMA rats. Overall excretion of XN and its metabolites within 48 h was only 4.6% of the ingested dose in GF rats and 4.2% in HMA rats, feces being the major route of excretion. While both GF and HMA rats excreted XN, IX, and their conjugates with urine and feces, 8-prenylnaringenin and its corresponding conjugates were exclusively observed in the feces of HMA rats. The microbial formation of 8-prenylnaringenin was confirmed by incubation of XN and IX with human fecal slurries. The amount of conjugates excreted in urine and feces was lower in HMA rats compared to GF rats indicating their hydrolysis by human intestinal microbiota. Thus, the impact of bacteria on the XN metabolism in the gut may affect the in vivo effects of ingested XN.

Long-term stability of the human gut microbiota in two different rat strains.

Human microbiota associated rats are frequently used as a model to study host microbe interactions. This study investigated the long-term stability of the bacterial community in such rats. Following the association of two strains of germ-free rats (12 male animals each) with fecal bacteria from a human donor the development of the microbiota was monitored for 12 months by PCR-denaturing gradient gel electrophoresis. During this time the Dice similarity coefficient (Cs) for the fecal microbial community of the rats associated with a human microbiota in comparison to the donor sample ranged between 73% +/- 8 and 74% +/- 3 for the Wistar and the Fischer 344 rats, respectively. After 12 months the similarity coefficients were 78% +/- 9 and 76% +/- 7, respectively, while the similarity coefficients for rat sample replicates ranged from 77% +/- 7 to 88% +/- 5; the similarity coefficient of the donor sample replicates was 78% +/- 9. DNA sequences of bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria previously found in samples of human, mouse or pig intestinal origin. The results of this study suggest that the dominant human fecal microbiota can be maintained in the human microbiota associated rat model for at least one year.

Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry.

Little is known about the occurrence and the fate of veterinary drugs in the environment. Therefore, a liquid chromatography/tandem mass spectrometry method was developed and employed to investigate in detail the distribution and persistence of the frequently used tetracyclines and tylosin in a field fertilized with liquid manure on April 2000 and April 2001; soil sampling was performed in May 2000, November 2000, and May 2001. We detected 4.0 mg/kg tetracycline and 0.1 mg/kg chlortetracycline in the liquid manure of April 2000, as well as comparable amounts in the liquid manure of April 2001. In the soil samples of May 2001, the highest average concentrations of 86.2 (0-10 cm), 198.7 (10-20 cm), and 171.7 microg/kg (20-30 cm) tetracycline and 4.6-7.3 micro/kg chlortetracycline (all three sublayers) were found. At soil depths between 30 and 90 cm, as well as in soil or groundwater, tetracyclines could not be detected. In addition, oxytetracycline and tylosin could not be detected in any sample investigated. We conclude that tetracyclines enter the environment in significant concentrations via repeated fertilizations with liquid manure, build up persistent residues, and accumulate in soil. Therefore, tetracyclines may have a potential risk and investigations on the environmental effects of these antibiotics are necessary.

Marker rescue studies of the transfer of recombinant DNA to Streptococcus gordonii in vitro, in foods and gnotobiotic rats.

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 x 10(-6) to 5.8 x 10(-7) transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 x 10(-9)). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 x 10(-10)), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 x 10(-11). No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.