Tetracycline Residues and Tetracycline Resistance Genes in Groundwater Impacted by Swine Production Facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.

To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators—including inorganic ions, antibiotics, and antibiotic resistance genes—were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 μg/L.

Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes—tet(M), tet(O), tet(Q), and tet(W)—were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Biochemical and microbiological evidence for fermentative digestion in free-living land iguanas (Conolophus pallidus) and marine iguanas (Amblyrhynchus cristatus) on the Galápagos archipelago

Herbivorous lizards are potentially capable of high digestive efficiency, but the presence of an indigenous microbial population has been implied from measurements of activity rather than directly studied. This study is the first to provide direct biochemical and microbiological evidence for fermentative digestion in free-living land iguanas (Conolophus pallidus) and marine iguanas (Amblyrhynchus cristatus) from the Galapagos archipelago. In marine iguanas, the stomach and large capacious colon contained ca. 32% and 60%, respectively, of the weight of total gut content. Total volatile fatty acid concentration was ca. 150 and 180 mM, respectively, for marine and land iguanas. Molar proportions of acetate, propionate, and butyrate (80.3%, 9.5%, and 3.5%) in land iguana fecal samples were similar to those for marine iguanas. Examination of fecal samples using confocal and transmission electron microscopy, as well as cultivable counts, revealed a dense and diverse population of bacteria, with spores prominent. Total culturable counts of anaerobes (2.22x10(8) g(-1) wet weight of fecal material) outnumbered aerobes on average by a factor of ca. 700. Combined, these results strongly support the contention that these unique herbivorous lizards are largely dependent on the presence and metabolic activities of a resident bacterial population in order to hydrolyze and ferment plant polymers that are indigestible to the host.     

Ecology of uncultivated Oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.     

Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria in human feces

16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces. Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals. Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study. Bacteria related to E. hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Importance of microbial colonization of the gut in early life to the development of immunity

The mammalian gastrointestinal tract harbors a complex microbiota consisting of between 500 and 1000 distinct microbial species. Comparative studies based on the germ-free gut have provided clear evidence that the gut microbiota is instrumental in promoting the development of both the gut and systemic immune systems. Early microbial exposure of the gut is thought to dramatically reduce the incidence of inflammatory, autoimmune and atopic diseases further fuelling the scientific viewpoint, that microbial colonization plays an important role in regulating and fine-tuning the immune system throughout life. Recent molecular diversity studies have provided additional evidence that the human gut microbiota is compositionally altered in individuals suffering from inflammatory bowel disorders, suggesting that specific bacterial species are important to maintaining immunological balance and health. New and exciting insights into how gut bacteria modulate the mammalian immune system are emerging. However, much remains to be elucidated about how commensal bacteria influence the function of cells of both the innate and adaptive immune systems in health and disease.     

Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale

Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation.