Authors: Torey Looft, Heather K Allen, Thomas A Casey...

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.

PubMed: 24959163
DOI: 10.3389/fmicb.2014.00276

Authors: Ahmed A Abouelkhair, Mohamed N Seleem

UNLABELLED

is an enteric pathogen that can cause a range of illnesses from mild diarrhea to pseudomembranous colitis and even death. This pathogen often takes advantage of microbial dysbiosis provoked by antibiotic use. With the increasing incidence and severity of infections, coupled with high recurrence rates, there is an urgent need to identify innovative therapies that can preserve the healthy state of the gut microbiota. In this study, we screened a microbial metabolite library against . From a collection of 527 metabolites, we identified 18 compounds with no previously identified antimicrobial activity and metabolites that exhibited potent activity against growth. Of these 18 hits, five drugs and three metabolites displayed the most potent anti-. activity and were subsequently assessed against 20 clinical isolates of . These potent agents included ecteinascidin 770 (minimum inhibitory concentration against 50% of isolates [MIC] ≤0.06 µg/mL); 8-hydroxyquinoline derivatives, such as broxyquinoline and choloroquinaldol (MIC = 0.125 µg/mL); ionomycin calcium salt, carbadox, and robenidine hydrochloride (MIC = 1 µg/mL); and dronedarone and milbemycin oxime (MIC = 4 µg/mL). Unlike vancomycin and fidaxomicin, which are the standard-of-care anti-. antibiotics, most of these metabolites showed robust bactericidal activity within 2-8 h with minimal impact on the growth of representative members of the normal gut microbiota. These results suggest that the drugs and microbial metabolite scaffolds may offer alternative avenues to address unmet needs in disease prevention and treatment.

IMPORTANCE

The most frequent infection associated with hospital settings is , which can cause fatal diarrhea and severe colitis, toxic megacolon, sepsis, and leaky gut. Those who have taken antibiotics for other illnesses that affect the gut's healthy microbiota are more susceptible to infection (CDI). Recently, some reports showed higher recurrence rates and resistance to anti-, which may compromise the efficacy of CDI treatment. Our study is significant because it is anticipated to discover novel microbial metabolites and drugs with microbial origins that are safe for the intestinal flora, effective against , and reduce the risk of recurrence associated with CDI.

Topics: Clostridioides difficile; Anti-Bacterial Agents; Microbial Sensitivity Tests; Humans; Clostridium Infections; Gastrointestinal Microbiome

PubMed: 38940508
DOI: 10.1128/msphere.00273-24

Authors: Yuan-Tai Hung, Qiong Hu, Richard J Faris...

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)-1β 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.

PubMed: 32751572
DOI: 10.3390/antibiotics9080463

Authors: Timothy A Johnson, Torey Looft, Andrew J Severin...

Carbadox is a quinoxaline-di--oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction ; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the 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 ( = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism ( < 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 ( ≤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. 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.

Topics: Animal Feed; Animals; Anti-Infective Agents; Bacteriophages; Carbadox; Drug Resistance, Microbial; Feces; Gastrointestinal Microbiome; Gene Expression Profiling; Metagenome; Prophages; Sus scrofa; Swine; Transcription, Genetic

PubMed: 28790203
DOI: 10.1128/mBio.00709-17

Authors: Heying Zhang, Wei Qu, Yanfei Tao...

This paper presents a convenient and sensitive LC-MS/MS method for the simultaneous determination of carbadox and olaquindox residues, including desoxyolaquindox (DOLQ), desoxycarbadox (DCBX), quinoxaline-2-carboxylic acid (QCA), 3-methyl-quinoxaline-2-carboxylic acid (MQCA), and the glycine conjugates of QCA and MQCA (namely, QCA-glycine and MQCA-glycine, resp.) in swine muscle and liver tissues. Tissue samples were extracted with 2% metaphosphoric acid in 20% methanol and cleaned up by solid-phase extraction (SPE) on a mixed-mode anion-exchange column (Oasis MAX). Analysis was performed on a C column by detection with mass spectrometry in the multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the six analytes were determined to be 0.01 g·kg to 0.25 g·kg, and the limits of quantification (LOQs) were 0.02 g·kg to 0.5 g·kg. The total recoveries of the six analytes in all tissues were higher than 79.1% with the RSD% less than 9.2%. The developed method can determine the real residue level of QCA and MQCA, whether they are present in free form or as glycine conjugates in tissues, together with the carcinogenic desoxy metabolites DCBX and DOLQ with high recovery. Therefore, this method was suitable for routine analysis of residue control programmes and the residue depletion study of CBX and OLQ on swine.

PubMed: 30079259
DOI: 10.1155/2018/2834049

Authors: Liudmila Kulakova, Andrey Galkin, Catherine Z Chen...

Giardiasis is a severe intestinal parasitic disease caused by Giardia lamblia, which inflicts many people in poor regions and is the most common parasitic infection in the United States. Current standard care drugs are associated with undesirable side effects, treatment failures, and an increasing incidence of drug resistance. As follow-up to a high-throughput screening of an approved drug library, which identified compounds lethal to G. lamblia trophozoites, we have determined the minimum lethal concentrations of 28 drugs and advanced 10 of them to in vivo studies in mice. The results were compared to treatment with the standard care drug, metronidazole, in order to identify drugs with equal or better anti-Giardia activities. Three drugs, fumagillin, carbadox, and tioxidazole, were identified. These compounds were also potent against metronidazole-resistant human G. lamblia isolates (assemblages A and B), as determined in in vitro assays. Of these three compounds, fumagillin is currently an orphan drug used within the European Union to treat microsporidiosis in immunocompromised individuals, whereas carbadox and tioxidazole are used in veterinary medicine. A dose-dependent study of fumagillin in a giardiasis mouse model revealed that the effective dose of fumagillin was ∼ 100-fold lower than the metronidazole dose. Therefore, fumagillin may be advanced to further studies as an alternative treatment for giardiasis when metronidazole fails.

Topics: Aminopeptidases; Animals; Antiprotozoal Agents; Axenic Culture; Carbadox; Cyclohexanes; Drug Discovery; Drug Resistance; Fatty Acids, Unsaturated; Giardia lamblia; Giardiasis; Glycoproteins; High-Throughput Screening Assays; Humans; Inhibitory Concentration 50; Methionyl Aminopeptidases; Metronidazole; Mice; Parasitic Sensitivity Tests; Sesquiterpenes; Species Specificity; Structure-Activity Relationship; Thiazoles; Trophozoites

PubMed: 25267663
DOI: 10.1128/AAC.03834-14

Authors: Jeferson M Lourenco, Rachel S Hampton, Hannah M Johnson...

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 ( = 0.04) in the group of piglets fed carbadox. Faith's phylogenetic diversity was similar ( = 0.89) for both groups of piglets on day 35, but it was diminished ( = 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 ( = 0.27) in both groups on day 63. Likewise, abundances of , and were all similar between the two groups ( ≥ 0.40) on day 35, but were smaller in the carbadox group ( ≤ 0.05) on day 49; however, on day 63, abundances of all these genera were once again similar ( ≥ 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 ( ≥ 0.52), but decreased in the carbadox group on day 49 ( ≤ 0.05), and were similar again in both groups on day 63 ( ≥ 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.

PubMed: 33778032
DOI: 10.3389/fvets.2021.601394

Authors: Lingling Guo, Xiaoling Wu, Gang Cui...

Abused or misused carbadox (CBX) and cyadox (CYA) in animal feed may cause food safety concerns, threatening human health. Here, we describe the design of a novel hapten for preparation of a monoclonal antibody against CBX and CYA simultaneously. Using this antibody, colloidal gold immunochromatographic assay (GICA) was developed for screening of CBX and CYA residues in chicken breast. Under optimal conditions, semiquantitative analysis results were visible by eye, with a visual limit of detection of 8 μg/kg for CBX and CYA, and cut-off values of 20 μg/kg for CBX and 40 μg/kg for CYA in chicken breast. Quantitative analysis could be performed using a hand-held strip scanner, with a calculated limit of detection of 2.92 μg/kg for CBX and 2.68 μg/kg for CYA in chicken breast. Validated by liquid chromatography-MS/MS, the developed GICA provides a useful tool for rapid on-site CBX and CYA residue screening in chicken breast.

PubMed: 32010814
DOI: 10.1021/acsomega.9b02931

Authors: Payton L Dahmer, Grace E Leubcke, Annie B Lerner...

The objective of this experiment was to evaluate the effects of medium-chain fatty acids (MCFA) on nursery pig performance in place of ZnO and carbadox. In this trial, 360 weanling pigs (DNA 200 × 400; 5.4 ± 0.07 kg BW) were fed for 35 d, with 6 pigs/pen and 10 replicate pens/treatment. Upon weaning, pigs were weighed and allotted to pens based on BW in a completely randomized design to one of six treatment diets: 1) Negative control (no added ZnO or carbadox); 2) Control + 3,000 ppm ZnO in phase 1 and 2,000 ppm ZnO in phase 2; 3) Control + 50 g/ton carbadox; 4) Control + C6:C8:C10 MCFA blend; 5) Control + Proprietary Oil Blend (Feed Energy Corp.); and 6) Control + monolaurate blend (FORMI GML from ADDCON). Treatment diets were fed through two dietary phases and a common diet fed through phase three. Pigs and feeders were individually weighed on a weekly basis to determine average daily gain (ADG) and average daily feed intake (ADFI). From days 0 to 19, pigs being fed the ZnO or Carbadox diets had the greatest ADG. These pigs had significantly higher ( < 0.05) ADG than pigs fed the control or Feed Energy Proprietary Oil Blend, whereas pigs fed the C6:C8:C10 blend or FORMI GML diets had similar ( > 0.05) ADG compared with those fed carbadox. These effects were primarily driven by feed intake, which was greatest ( < 0.05) in pigs fed ZnO and carbadox. Treatment diet had a marginally significant effect ( = 0.078) on G:F. Increased day 19 BW ( < 0.05) was observed for pigs fed ZnO and carbadox compared with the negative control, whereas other treatments were intermediate. Additionally, blood data and fecal scores were collected throughout the trial. On day 21, pigs fed ZnO or carbadox had higher ( < 0.0001) glucose values than those fed the Feed Energy Proprietary Oil Blend, with other diets being intermediate, showing potential health benefits of carbadox. Although ZnO resulted in higher glucose values, it may also contribute to hepatic issues. Although replacing ZnO and carbadox with MCFA did not result in significant changes in gut microflora, it did affect fecal consistency by softening the feces during the treatment period. Overall, these results show that ZnO and carbadox are valuable additives to help maximize growth performance in early stages of the nursery. Some MCFA products, like FORMI GML, may result in similar performance, whereas others restrict it. Thus, additional research is needed to study the effectiveness of MCFA to replace ZnO or feed-based antibiotics.

PubMed: 32939444
DOI: 10.1093/tas/txaa151

Authors: Cynthia Jinno, Xunde Li, Yanhong Liu...

Our previous research reported that supplementation of DSM 25841 promoted growth and disease resistance of weaned pigs under enterotoxigenic (ETEC) challenge and its efficacy is comparable to carbadox. This follow-up study aimed to characterize the effects of ETEC infection, supplementing DSM 25841 or carbadox on intestinal microbiota of pigs. Forty-eight weaned pigs (6.17 ± 0.36 kg BW) were randomly allotted to one of four treatments: negative control (NC), positive control (PC), antibiotics (AGP, 50 mg/kg of carbadox), and direct fed microbials (DFM, 2.56 × 10  CFU/kg of ). The experiment lasted 28 days with 7 days before and 21 days after first inoculation (day 0). Pigs in the PC, AGP, and DFM groups were orally inoculated with F18 ETEC for 3 consecutive days with 10  CFU per dose per day. Fecal samples were collected on day -7, and day 7 and day 21 post inoculation, digesta samples were collected from jejunum, ileum, and distal colon on day 21 post inoculation to perform 16S rRNA sequencing. Sampling days and locations influenced ( < 0.05) Chao1 index and beta-diversity. Age increased (  < 0.05) the relative abundance of Firmicutes but decreased ( < 0.05) the relative abundance of Bacteroidetes in feces. ETEC infection increased (  < 0.05) the relative abundance of Proteobacteria in feces on day 7 post inoculation. AGP reduced ( < 0.05) relative abundance of Firmicutes and in feces compared with PC and DFM. AGP reduced ( < 0.05) relative abundance of in jejunum and ileum, while DFM reduced ( < 0.05) relative abundance of in jejunum and in ileum, compared with PC. Pigs fed with DFM had greater ( < 0.05) relative abundance of , , in jejunum, in ileum and colon, and in colon than pigs in AGP. Current results indicate that carbadox or had stronger influences on microbial diversity and composition in ileum than other intestinal segments and feces. Supplementation of could increase or maintain the relative abundance of beneficial bacteria in ileum compared with carbadox.

PubMed: 36620005
DOI: 10.3389/fmicb.2022.1064328