Impact of an oligosaccharide-based polymer on the metabolic profiles and microbial ecology of weanling pigs experimentally infected with a pathogenic E. coli.

BACKGROUND: Our previous study has reported that supplementation of oligosaccharide-based polymer enhances gut health and disease resistance of pigs infected with enterotoxigenic E. coli (ETEC) F18 in a manner similar to carbadox. The objective of this study was to investigate the impacts of oligosaccharide-based polymer or antibiotic on the host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18. RESULTS: Multivariate analysis highlighted the differences in the metabolic profiles of serum and colon digesta which were predominantly found between pigs supplemented with oligosaccharide-based polymer and antibiotic. The relative abundance of metabolic markers of immune responses and nutrient metabolisms, such as amino acids and carbohydrates, were significantly differentiated between the oligosaccharide-based polymer and antibiotic groups (q < 0.2 and fold change > 2.0). In addition, pigs in antibiotic had a reduced (P < 0.05) relative abundance of Lachnospiraceae and Lactobacillaceae, whereas had greater (P < 0.05) Clostridiaceae and Streptococcaceae in the colon digesta on d 11 post-inoculation (PI) compared with d 5 PI. CONCLUSIONS: The impact of oligosaccharide-based polymer on the metabolic and microbial profiles of pigs is not fully understood, and further exploration is needed. However, current research suggest that various mechanisms are involved in the enhanced disease resistance and performance in ETEC-challenged pigs by supplementing this polymer.

Trace amounts of antibiotic altered metabolomic and microbial profiles of weaned pigs infected with a pathogenic E. coli.

BACKGROUND: Our previous study has shown that supplementation of trace amounts of antibiotic exacerbated the detrimental effects of enterotoxigenic E. coli (ETEC) infection and delayed the recovery of pigs that may be associated with modified metabolites and metabolic pathways. Therefore, the objective of this study was to explore the impacts of trace levels of antibiotic (carbadox) on host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18. RESULTS: The multivariate analysis highlighted a distinct metabolomic profile of serum and colon digesta between trace amounts of antibiotic (TRA; 0.5 mg/kg carbadox) and label-recommended dose antibiotic (REC; 50 mg/kg carbadox) on d 5 post-inoculation (PI). The relative abundance of metabolomic markers of amino acids, carbohydrates, and purine metabolism were significantly differentiated between the TRA and REC groups (q < 0.2). In addition, pigs in REC group had the highest (P < 0.05) relative abundance of Lactobacillaceae and tended to have increased (P < 0.10) relative abundance of Lachnospiraceae in the colon digesta on d 5 PI. On d 11 PI, pigs in REC had greater (P < 0.05) relative abundance of Clostridiaceae compared with other groups, whereas had reduced (P < 0.05) relative abundance of Prevotellaceae than pigs in control group. CONCLUSIONS: Trace amounts of antibiotic resulted in differential metabolites and metabolic pathways that may be associated with its slow responses against ETEC F18 infection. The altered gut microbiota profiles by label-recommended dose antibiotic may contribute to the promotion of disease resistance in weaned pigs.

Hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion extraction of carbadox and olaquindox in feeds.

BACKGROUND: Carbadox and olaquindox have been banned from feeds since 1998 by the EU because of their mutagenic, photoallergic, and carcinogenic effects. Unfortunately, owing to their outstanding effect, they are frequently abused or misused in animal husbandry. There is an urgent need to develop a sensitive and reliable method for monitoring these drugs in animal feeds. RESULTS: This work reported a new method of hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion (MMSPD) extraction coupled with reversed-phase liquid chromatography-mass spectrometry for simultaneous determination of carbadox and olaquindox in animal feeds. 3-Trimethoxysilylpropyl methacrylate (γ-MAPS)-modified attapulgite (ATP) was crosslinked with γ-MAPS-modified iron(II,III) oxide (Fe3 O4 ), 1-vinyl-3-(butyl-4-sulfonate) imidazolium (VBSIm), acrylamide (AM), and N,N'-methylene-bis(acrylamide) (MBA) to synthesize ATP@Fe3 O4 @poly(VBSIm-AM-MBA) particles. The resultant particles were characterized by scanning electron microscopy, energy dispersive spectrometer, transmission electron microscopy, vibrating sample magnetometer, and Fourier transform infrared spectroscopy. Crosslinking of ATP into the magnetic particles has significantly increased the adsorption capacity of the particles. Under optimum conditions, the limits of detection (S/N = 3) were 0.3 µg kg-1 and 0.9 µg kg-1 for carbadox and olaquindox respectively. The intra-day and inter-day recoveries of the spiked targets in feed samples were in the range 83.5-98.3% with relative standard deviations of 1.0-8.3%. CONCLUSION: With a simplified procedure and a low amount of sample, the proposed hydrophilic-interaction-based MMSPD method is not only useful for the determination of carbadox and olaquindox in feeds but also holds great promise for the analysis of other polar targets in solid or semisolid matrices. © 2021 Society of Chemical Industry.

The Effects of Feeding Antibiotic on the Intestinal Microbiota of Weanling Pigs.

This study investigated the use of carbadox in the diet of nursery pigs. Ten pens of weanling piglets were assigned to 2 treatments: one containing carbadox and another without it. From days 21 to 35 of age, the first group of piglets was fed carbadox at 55 mg/kg of diet; followed by 27.5 mg/kg from days 36 to 49; and 0 mg/kg from days 50 to 63. The second group of pigs was fed a control diet without carbadox from days 21 to 63 of age. On days 35, 49, and 63, fecal samples were collected directly from the rectum of 2 piglets in each pen, and the samples were subjected to microbial DNA sequencing and metagenomic functional analysis using the 16S rRNA gene. Feed conversion from days 21 to 63 was improved (P = 0.04) in the group of piglets fed carbadox. Faith's phylogenetic diversity was similar (P = 0.89) for both groups of piglets on day 35, but it was diminished (P = 0.01) in the carbadox-fed group on day 49; however, following the complete removal of carbadox from their diets, this microbial diversity index was once again found to be similar (P = 0.27) in both groups on day 63. Likewise, abundances of Slackia, Peptococcus, Catenibacterium, Coprococcus, and Blautia were all similar between the two groups (P ≥ 0.40) on day 35, but were smaller in the carbadox group (P ≤ 0.05) on day 49; however, on day 63, abundances of all these genera were once again similar (P ≥ 0.29). Metabolic pathways involved in cellular growth, death, and genetic information processing (translation) were found to be similarly expressed in the microbiota of piglets from both groups on day 35 (P ≥ 0.52), but decreased in the carbadox group on day 49 (P ≤ 0.05), and were similar again in both groups on day 63 (P ≥ 0.51). These results revealed that feeding carbadox to piglets during the first 4 weeks after weaning significantly affected their fecal microbiotas; however, 2 weeks after the removal of carbadox, those changes tended to disappear, indicating that the shifts were carbadox-dependent.

Trace amounts of antibiotic exacerbated diarrhea and systemic inflammation of weaned pigs infected with a pathogenic Escherichia coli.

The experiment was conducted to investigate the effects of trace amounts of antibiotic on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli. Weaned pigs (n = 34, 6.88 ± 1.03 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of the three dietary treatments: nursery basal diet (CON) and two additional diets supplemented with 0.5 or 50 mg/kg carbadox to the nursery basal diet (TRA or REC), respectively. The experiment lasted 18 d with 7 d before and 11 d after the first E. coli inoculation. The E. coli F18 inoculum was orally provided to all pigs with a dose of 1010 colony-forming unit (CFU)/3 mL for three consecutive days. Fecal and blood samples were collected on day 0 before inoculation and days 2, 5, 8, and 11 postinoculation (PI) to test the percentage of ß-hemolytic coliforms in total coliforms and complete blood cell count, respectively. Sixteen pigs were euthanized on day 5 PI, whereas the remaining pigs were euthanized at the end of the experiment to collect the jejunal and ileal mucosa and mesenteric lymph node for gene expression and bacterial translocation, respectively. Pigs in REC had greater (P < 0.05) final BW and lower (P < 0.05) overall frequency of diarrhea compared with pigs in the CON and TRA groups. Pigs in TRA had the lowest (P < 0.05) average daily gain and feed efficiency from day 0 to 5 PI, highest (P < 0.05) percentage of ß-hemolytic coliforms in fecal samples on days 2 and 5 PI, and greatest (P < 0.05) bacterial colonies in mesenteric lymph nodes on day 11 PI compared with pigs in the CON and REC groups. Pigs in TRA had the greatest (P < 0.05) neutrophils on day 5 PI and higher (P < 0.05) white blood cell counts and lymphocytes than other groups on day 11 PI. Pigs in TRA had the greatest (P < 0.05) serum C-reactive protein on days 2 and 5 PI and serum tumor necrosis factor-α on day 5 PI, compared with pigs in the CON and REC groups. Pigs fed REC had increased (P < 0.05) mRNA expression of zona occludens-1 (ZO-1) and occludin (OCDN) and reduced (P < 0.05) interleukin-1 beta (IL1B), interleukin-6 (IL6), and tumor necrosis factor-alpha (TNFA) in ileal mucosa on day 5 PI, compared with the CON, whereas TRA upregulated (P < 0.05) mRNA expression of IL1B, IL6, and cyclooxygenase-2 (COX2) in the ileal mucosa on day 11 PI, compared with the REC. In conclusion, trace amounts of antibiotic may exacerbate the detrimental effects of E. coli infection on pig performance by increasing diarrhea and systemic inflammation of weanling pigs.

Metabolomic Profile of Weaned Pigs Challenged with E. coli and Supplemented with Carbadox or Bacillus subtilis.

This study explored the metabolomic profiles in ileal mucosa and colon digesta in response to enterotoxigenic Escherichia coli F18 (ETEC) infection and dietary use of probiotics and low-dose antibiotics. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight) were randomly allotted to one of four treatments. Pigs in the negative control (NC) were fed a basal diet without ETEC challenge, whereas pigs in the positive control (PC), antibiotic, and probiotic groups were fed the basal diet, basal diet supplemented with 50 mg/kg of carbadox, or 500 mg/kg of Bacillus subtilis, respectively, and orally challenged with ETEC F18. All pigs were euthanized at day 21 post-inoculation to collect ileal mucosa and colon digesta for untargeted metabolomic profiling using gas chromatography coupled with time-of-flight mass spectrometry. Multivariate analysis highlighted a more distinct metabolomic profile of ileal mucosa metabolites in NC compared to the ETEC-challenged groups. The relative abundance of 19 metabolites from the ileal mucosa including polyamine, nucleotide, monosaccharides, fatty acids, and organic acids was significantly different between the NC and PC groups (q < 0.1). In colon digesta, differential metabolites including 2-monoolein, lactic acid, and maltose were reduced in the carbadox group compared with the probiotics group. In conclusion, several differential metabolites and metabolic pathways were identified in ileal mucosa, which may suggest an ongoing intestinal mucosal repair in the ileum of ETEC-challenged pigs on day 21 post-inoculation.

Analysis of Gastrointestinal Responses Revealed Both Shared and Specific Targets of Zinc Oxide and Carbadox in Weaned Pigs.

Antibiotics and pharmacological zinc supplementation were commonly used as growth promoters for several decades in the swine industry before being limited because of public health and environmental concerns. Further, the physiological and metabolic responses associated with their growth promotion effects are unclear. To characterize these responses induced by pharmacological zinc supplementation (2500 mg/kg) and carbadox (55 mg/kg), 192 post-weaning pigs were fed basal and test diets for 43 days. Compared with basal, pharmacological zinc and carbadox independently improved growth performance. Pharmacological zinc increased gastric mucosa thickness compared with basal zinc, while carbadox increased intestinal villus:crypt ratio compared with non-carbadox. Pharmacological zinc and carbadox independently reduced interleukin (IL)-1ß concentration compared with basal zinc and non-carbadox. Pharmacological zinc increased IL-1RA:IL-1 ratio by 42% compared with basal zinc, while carbadox tended to increase the IL-10 and IL10:IL-12 ratio compared with non-carbadox. Carbadox increased fecal concentrations of histidine and lysine compared with non-carbadox. The independent effect of pharmacological zinc and carbadox on morphology and nutrient metabolism, and their shared effect on immunity may contribute to the additive effect on growth promotion. These results further confirmed the concept that growth promotion is multifactorial intervention. Therefore, elucidating growth-promoting effects and searching for alternatives should include wide-spectrum evaluation.

Novel Electrochemical Sensor Fabricated for Individual and Simultaneous Ultrasensitive Determination of Olaquindox and Carbadox Based on MWCNT-OH/CMK-8 Hybrid Nanocomposite Film.

A hybrid nanocomposite consisting of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) and cube mesoporous carbon (CMK-8) was applied in this study to construct an MWCNT-OH/CMK-8/gold electrode (GE) electrochemical sensor and simultaneously perform the electro-reduction of olaquindox (OLA) and carbadox (CBX). The respective peak currents of CBX and OLA on the modified electrode increased by 720- and 595-fold relative to the peak current of GE. The performances of the modified electrode were investigated with electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry. Then, the modified electrodes were used for the individual and simultaneous determination of OLA and CBX. The fabricated sensor demonstrated a linear response at 0.2-500 nmol/L in optimum experimental conditions, and the detection limits were 104.1 and 62.9 pmol/L for the simultaneous determination of OLA and CBX, respectively. As for individual determination, wide linear relationships were obtained for the detected OLA with levels of 0.05-500 nmol/L with LOD of 20.7 pmol/L and the detected CBX with levels of 0.10-500 nmol/L with LOD of 50.2 pmol/L. The fabricated sensor was successfully used in the independent and simultaneous determination of OLA and CBX in spiked pork samples.

Determination of olaquindox, carbadox and cyadox in animal feeds by ultra-performance liquid chromatography tandem mass spectrometry.

Olaquindox, carbadox, and cyadox are chemically synthesised antibacterial and growth-promoting agents for animals. At high doses they may exert mutagenicity and hepatic and adrenal toxicities in animals. Regrettably, these substances are frequently abused or misused when added into animal feeds. Thus, developing a sensitive and reliable method for simultaneous determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds is crucially important for food safety monitoring. In this paper we optimised instrumental conditions, extraction solvents, solid phase extraction cartridges, and pH of the loading solvents on the Oasis HLB cartridge. Under the optimal conditions, mean recoveries ranged from 74.1 to 111%, and intra-day and inter-day variations were lower than 14.6% and 10.8%, respectively. The limits of quantification for olaquindox, carbadox, and cyadox were 0.05 mg kg-1, 0.10 mg kg-1, and 0.025 mg kg-1, respectively. The proposed method uses ultra-performance liquid chromatography tandem mass spectrometry and is sensitive and reliable for the simultaneous determination of olaquindox, carbadox, and cyadox in three kinds of animal feeds (specifically, mixed feed, concentrated feed, and additive premixed feed). This method has good precision, high sensitivity, and good reproducibility, and thus it can be used for convenient and accurate determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds.

Bacterial Whack-a-Mole: Reconsidering the Public Health Relevance of Using Carbadox in Food Animals.

Carbadox is an antibiotic used to control dysentery and promote growth in swine in the United States; however, the drug also causes tumors and birth defects in laboratory animals. Despite this and because the drug has no analogs in human medicine, it is not considered "medically important" and can be used in livestock without veterinarian oversight. In their recent study, T. A. Johnson et al. (mBio 8:e00709-17, 2017, https://doi.org/10.1128/mBio.00709-17) demonstrated that carbadox has profound effects on the swine gut microbiome, including the induction of transducing phage carrying tetracycline, aminoglycoside, and beta-lactam resistance genes. In swine production, carbadox can be used in conjunction with other antibiotics (e.g., oxytetracycline) that could fuel the emergence of strains carrying phage-encoded resistance determinants. Johnson et al.'s findings underscore the potential unforeseen consequences of using antibiotics in livestock production and call into question our current methods for classifying whether or not a veterinary drug has relevance to human health.

The In-Feed Antibiotic Carbadox Induces Phage Gene Transcription in the Swine Gut Microbiome.

Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo longitudinal effects of carbadox on swine gut microbial gene expression (fecal metatranscriptome) and phage population dynamics (fecal dsDNA viromes). Microbial metagenome, transcriptome, and virome sequences were annotated for taxonomic inference and gene function by using FIGfam (isofunctional homolog sequences) and SEED subsystems databases. When the beta diversities of microbial FIGfam annotations were compared, the control and carbadox communities were distinct 2 days after carbadox introduction. This effect was driven by carbadox-associated lower expression of FIGfams (n = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism (q < 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FIGfams related to all stages of the phage lytic cycle 2 days following the introduction of carbadox (q ≤0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. These effects were diminished by 7 days of continuous carbadox in the feed, suggesting an acute impact. Additionally, the viromes included a few genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics but these did not change in frequency over time or with treatment. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes in swine gut bacterial communities as a result of carbadox administration.IMPORTANCE FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Interestingly, the present study shows greater prophage gene expression in feces from carbadox-fed animals than in feces from nonmedicated animals 2 days after the initiation of in-feed carbadox treatment. Importantly, the phage genetic material isolated in this study contained genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered.

The critical role of oxidative stress in the toxicity and metabolism of quinoxaline 1,4-di-N-oxides in vitro and in vivo.

Quinoxaline 1,4-dioxide derivatives (QdNOs) have been widely used as growth promoters and antibacterial agents. Carbadox (CBX), olaquindox (OLA), quinocetone (QCT), cyadox (CYA) and mequindox (MEQ) are the classical members of QdNOs. Some members of QdNOs are known to cause a variety of toxic effects. To date, however, almost no review has addressed the toxicity and metabolism of QdNOs in relation to oxidative stress. This review focused on the research progress associated with oxidative stress as a plausible mechanism for QdNO-induced toxicity and metabolism. The present review documented that the studies were performed over the past 10 years to interpret the generation of reactive oxygen species (ROS) and oxidative stress as the results of QdNO treatment and have correlated them with various types of QdNO toxicity, suggesting that oxidative stress plays critical roles in their toxicities. The major metabolic pathways of QdNOs are N→O group reduction and hydroxylation. Xanthine oxidoreductase (XOR), aldehyde oxidase (SsAOX1), carbonyl reductase (CBR1) and cytochrome P450 (CYP) enzymes were involved in the QdNOs metabolism. Further understanding the role of oxidative stress in QdNOs-induced toxicity will throw new light onto the use of antioxidants and scavengers of ROS as well as onto the blind spots of metabolism and the metabolizing enzymes of QdNOs. The present review might contribute to revealing the QdNOs toxicity, protecting against oxidative damage and helping to improve the rational use of concurrent drugs, while developing novel QdNO compounds with more efficient potentials and less toxic effects.

HPLC-MS/MS method validation for the detection of carbadox and olaquindox in poultry and swine feedingstuffs.

Carbadox (CBX) and olaquindox (OLA) were used in poultry and swine feed for growth promotion, to improve feed efficiency and increase the rate of weight gain. However, the use of these agents in feedingstuffs was prohibited because of concerns about their toxicity. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban. A quantitative and confirmatory method for determining the presence of CBX and OLA in poultry and swine feed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed, optimized, and validated. The analytes extraction was performed with a mixture of water and acetonitrile (1:1v/v) and cleanup with hexane and C18 (dispersive phase). The method was evaluated by the following parameters: specificity, linearity, matrix effect, decision limits (CCα), detection capability (CCß), accuracy, precision, limits of detection (LoD), limits of quantification (LoQ) and measurement uncertainty. The validated method presented a broad linear study range and no significant matrix effect. The limit of detection (LoD) was defined at 9 µg kg(-1) for CBX and 80 µg kg(-1) for OLA, and the limit of quantification (LoQ) was defined at 12 µg kg(-1) and 110 µg kg(-1) for CBX and OLA, respectively. The accuracy of the method was adequate for CBX and OLA. The recovery values found in the repeatability conditions were 99.41% for CBX and 104.62% for OLA. Under intralaboratory reproducibility conditions, the values were 98.63% for CBX and 95.07% for OLA. It was concluded that the performance parameters demonstrated total method adequacy for the detection and quantification of CBX and OLA in poultry and swine feedingstuffs.

In vivo studies to highlight possible illegal treatments of rabbits with carbadox and olaquindox.

For the treatment of rabbit dysentery and bacterial enteritis, veterinary practitioners often adopt veterinary medicinal products authorised for other food-producing species, but in some cases non-authorised drugs frequently used in the past, such as carbadox and olaquindox, might be illegally adopted. To verify the carbadox and olaquindox distribution and persistence in rabbit tissues, two independent in vivo studies were carried out. In the first study, 24 healthy rabbits received water medicated with carbadox at 100 mg l(-1) over a period 28 days, whereas in the second one, 24 healthy rabbits were administered water containing olaquindox at 100 mg l(-1). In each study rabbits were randomly assigned to four groups to be sacrificed respectively at 0, 5, 10 and 20 days from treatment withdrawal, for depletion studies. A control group of six animals was adopted for control and as a reservoir of blank tissues. Muscle and liver samples collected from each treated animal were stored at -20°C pending the analysis. Sensitive and robust liquid chromatography-tandem mass spectrometry analytical methods were set up for the parent compounds and their main metabolites quinoxaline-2-carboxylic acid, desoxycarbadox and 3-methylquinoxaline-2-carboxylic acid to verify their residual. Data collected demonstrate that the combination of liver as target matrix, quinoxaline-2-carboxylic acid and 3-methylquinoxaline-2-carboxylic acid as marker residue and enzymatic digestion is strategic to evidence carbadox and/or olaquindox illegal treatments in rabbits, even 20 days after treatment withdrawal at concentration levels higher than 0.5 µg kg(-1). This findings suggests that liver should be proposed as target matrix for official control in national monitoring plan.

Detecting quinoxaline-2-carboxylic acid in animal tissues by using sensitive rapid enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay.

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluoroimmunoassay (TR-FIA) based on an anti-N-butylquinoxaline-2-carboxamide (BQCA) monoclonal antibody were standardized and validated for quinoxaline-2-carboxylic acid (QCA) screening in animal tissues and its performance were compared to HPLC. The sensitivities obtained for edible tissue extracts were 1.62 and 1.12 ng ml(-1) for ic-ELISA and TR-FIA detection, respectively. Two samples were spiked with QCA and analyzed by both methods. The recovery values ranged from 92.6% to 112.2% and the coefficients of variation were less than 15% for QCA spiking into swine tissue samples at concentrations of 2.5-50.0 µg kg(-1). Excellent correlations (r(2)=0.987-0.996) of the ic-ELISA/HPLC and TR-FIA/HPLC data were observed for processed samples. The results demonstrated that the ic-ELISA and TR-FIA methods were rapid and accurate for the residue detection of QCA in animal tissues.

Carbadox has both temporary and lasting effects on the swine gut microbiota.

Antibiotics are used in livestock and poultry production to treat and prevent disease as well as to promote animal growth. Carbadox is an in-feed antibiotic that is widely used in swine production to prevent dysentery and to improve feed efficiency. The goal of this study was to characterize the effects of carbadox and its withdrawal on the swine gut microbiota. Six pigs (initially 3-weeks old) received feed containing carbadox and six received unamended feed. After 3-weeks of continuous carbadox administration, all pigs were switched to a maintenance diet without carbadox. DNA was extracted from feces (n = 142) taken before, during, and following (6-week withdrawal) carbadox treatment. Phylotype analysis using 16S rRNA sequences showed the gradual development of the non-medicated swine gut microbiota over the 8-week study, and that the carbadox-treated pigs had significant differences in bacterial membership relative to non-medicated pigs. Enumeration of fecal Escherichia coli showed that a diet change concurrent with carbadox withdrawal was associated with an increase in the E. coli in the non-medicated pigs, suggesting that carbadox pre-treatment prevented an increase of E. coli populations. In-feed carbadox caused striking effects within 4 days of administration, with significant alterations in both community structure and bacterial membership, notably a large relative increase in Prevotella populations in medicated pigs. Digital PCR was used to show that the absolute abundance of Prevotella was unchanged between the medicated and non-medicated pigs despite the relative increase shown in the phylotype analysis. Carbadox therefore caused a decrease in the abundance of other gut bacteria but did not affect the absolute abundance of Prevotella. The pending regulation on antibiotics used in animal production underscores the importance of understanding how they modulate the microbiota and impact animal health, which will inform the search for antibiotic alternatives.

The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella.

Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the environment.

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry.

This paper presents LC-MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3-methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0µgkg(-1). Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45°C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexION™ technology to reduce matrix effect was used. The decision limit (CCα) ranged from 1.04µgkg(-1) to 2.11µgkg(-1) and the detection capability (CCß) ranged from 1.46µgkg(-1) to 2.89µgkg(-1). The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC.

Genotoxicity of quinocetone, cyadox and olaquindox in vitro and in vivo.

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His(+) mutants at 6.9 µg/plate in Salmonella typhimurium TA 97, at 18.2 µg/plate in TA 100, TA 1535, TA 1537, and at 50 µg/plate in TA 98. CYA produced His(+) mutants at 18.2 µg/plate in TA 97, TA 1535, and at 50 µg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ≥10 µg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.