Bridging of a Liquid Chromatography-Tandem Mass Spectrometry Method for Oxytetracycline, Chlortetracycline, and Tetracycline in Bovine Kidney with the Official Microbial Growth Inhibition Assay.

BACKGROUND: Oxytetracycline (OTC), chlortetracycline (CTC), and tetracycline (TC) are approved antibiotics used to treat bacterial infections in cattle. To ensure human food safety, a tolerance has been established for the sum of these three TC residues as 12 parts per million in bovine kidney in the United States The current official regulatory method for quantifying these antibiotics in the target organ is a labor-intensive microbiological assay. OBJECTIVE: Our laboratory developed and validated a fast, selective, and less laborious method utilizing LC-tandem mass spectrometry for the determination and confirmation of the three tetracyclines (TET) in bovine kidney. METHODS: Briefly, homogenized kidney tissue was spiked with an internal standard (ISTD), and then was extracted with 1% phosphate buffer. The crude extract was cleaned up using solid-phase extraction cartridges before instrumental analysis. RESULTS: Accuracies for quantifying these three drugs in fortified kidney homogenate were between 99.9 and 110% at multiple concentrations, with respective CVs all below 9.5%. Quantitative correlation between the two methods (bridging) was evaluated with incurred bovine kidney samples for each of the three tetracyclines separately. The results were statistically evaluated using a measurement model called Functional Relationship Estimation by Maximum Likelihood. CONCLUSION: A linear quantitative relationship was demonstrated between the two methods within the concentration range of regulatory relevance. HIGHLIGHTS: This instrumental method is in addition to the established microbial assay for the detection of tetracyclines residue in beef kidney to ensure the food safety of cattle products.

A microbial endocrinology-based simulated small intestinal medium for the evaluation of neurochemical production by gut microbiota.

Microbial endocrinology represents the union of microbiology and neurobiology and is concerned with the ability of neurochemicals to serve as an evolutionary-based language between host and microbiota in health and disease. The recognition that microorganisms produce, modify and respond to the same neurochemicals utilized in the various signaling pathways of their mammalian hosts is increasingly being recognized as a mechanism by which the host and microbiota may interact to influence the progression of infectious disease as well as influence behavior through the microbiota-gut-brain axis. While the capacity for bacteria to produce neurochemicals has been recognized for decades, the degree to which this occurs in the environment of the gastrointestinal tract is still poorly understood. By combining techniques used in analytic chemistry, food science and environmental microbiology, a novel culture-based method was developed which generates a medium utilizing animal feed which resembles the contents of the small intestine. The usage of this medium allows for the in vitro growth of bacteria native to the gastrointestinal tract in an environment that is reflective of the small-intestinal host-based milieu. We describe a detailed protocol for the preparation of this medium and the quantification of neurochemicals by microorganisms grown therein. Catecholamines including dopamine and its precursor L-3,4-dihydroxyphenalanine (L-DOPA) as well as biogenic amines including tyramine and its precursor tyrosine, serve as prototypical examples of neurochemicals that are quantifiable with the methods described herein.

Determination of the concentration of potential efflux pump inhibitors, pheophorbide a and pyropheophorbide a, in the feces of animals by fluorescence spectroscopy.

Efflux pumps are vital bacterial components, and research has demonstrated that some plant compounds such as pheophorbide a (php) possess efflux pump inhibitor (EPI) activity. This study determined the quantity of php present in feces as an indicator of EPI activity. Feces were collected from different species of animals fed a variety of feeds. The chlorophyll metabolites php and pyropheophorbide a (pyp) were determined using fluorescense spectroscopy. The average concentrations [µg/g dry matter (DM) feces] of pyp/php in feces were as follows: guinea pig, 180; goat, 150; rabbit, 150; dairy cow, 120; feedlot cattle, 60; rat, <1; pig, <1; chicken, <1. These data indicate that animals consuming "green" diets will excrete feces with concentrations of php/pyp that exceed levels demonstrated to be inhibitory to bacterial efflux pumps (0.5 µg/mL). The natural presence EPIs in the gastrointestinal tract may modulate the activity of microbial efflux pumps and exert selection pressure upon resident microbial populations.