Microbial screening methods for detection of antibiotic residues in slaughter animals.
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ABSTRACT:
SUBMITTER: Pikkemaat MG
PROVIDER: S-EPMC2758187 | biostudies-other | 2009 Oct
REPOSITORIES: biostudies-other
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Project description:Even though antibiotics are necessary in livestock production, they can be harmful not only due to their toxicity, but also in view of their contribution to the emergence of antimicrobial resistance. Screening tests based on microbial growth inhibition appeared to be useful tools to prevent its entry into the food chain. They have nevertheless been traditionally carried out post mortem, leading to great economical loss and harm to the environment in case a positive sample is found. Hence, the objective was to evaluate the use of a screening test as an ante mortem alternative for the detection of antibiotic residues in meat: thus, Explorer®-Blood test was optimized and validated. After adapting the procedure for matrix preparation, the assay parameters were assessed from 344 antibiotic-free blood serum samples. Limits of Detection (LoDs) were defined by spiking blood serum with several of the most common antimicrobials used in veterinary practice. LoDs were similar to those obtained for meat and were at or below the maximum residue limits set by EU legislation for muscle. Analyses of in vivo injected samples, previously characterized by LC-MS/MS, demonstrated the method's accuracy and proved that Explorer®-Blood can be considered a suitable alternative to conventional post mortem screening methods.
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Project description:tylosin antibiotic fermentation residues Raw sequence reads
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Project description:Objective:To analyze the presence of selective antibiotic residues (oxytetracycline, amoxicillin, and ciprofloxacin) in milk during the antibiotic treatment course, and to evaluate the thermal effect on antibiotics residual status in milk of antibiotic-treated cows. Materials and Methods:The raw fresh milk was collected from 18 lactating cows before antibiotics treatment, which were brought to the veterinary hospital and suffered from either mastitis, foot and mouth disease, fever, local wound, or non-specific diarrhea, and so on. Out of the 18 lactating cows, six were treated with oxytetracycline, six were treated with amoxicillin, and six were treated with ciprofloxacin parenterally. Milk samples were also collected at 2nd day during treatment and final collection was done after maintaining the withdrawal period. Since milk is heated before consumption, it was boiled at 100°C for 20 min to evaluate the thermal effect on antibiotics residual status. Thin-layer chromatography was done for screening of antibiotics residue before and after boiling of the milk. Results:At day 0 (before antibiotic treatment), no antibiotics (oxytetracycline, ciprofloxacin and amoxicillin) residue was detected in raw milk of antibiotic treated cows. In contrast, on day 2 (during antibiotic treatment), 100% raw milk samples showed positive for antibiotics residue. After boiling, all milk samples showed positive for such specific antibiotics residue. On the other hand, no antibiotics residues were detected on day 9, which indicates the completion of the withdrawal period of the respective antibiotic. The intensities of bands for antibiotic on thin-layer chromatography plate of antibiotic residues in milk samples (oxytetracycline, amoxicillin, and ciprofloxacin) expressed that the respective antibiotic residual status was higher in the boiled milk compare to the raw milk. Conclusion:Proper maintenance of withdrawal period after antibiotic treatment would minimize the risk of antibiotic residues in milk, and boiling does not change these specific antibiotics residual status in milk. Therefore, awareness regarding the proper maintenance of withdrawal period after antibiotic treatment in lactating cows is one of the best strategies that may positively reduce the risk of antimicrobial drugs residue in milk.
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Project description:In the present study, antibiotic residues were detected in milk samples collected from the dairy herds located in Karnataka, India, by microbiological assay. Subsequently, the detected antibiotics were identified as azithromycin and tetracycline, by high-performance liquid chromatography, further both the antibiotics detected in the cow milk samples were found to be at high concentration (9708.7 and 5460 μg kg-1, respectively). We then investigated the effects of temperature and pH on the stabilities of azithromycin and tetracycline to determine the degradation rate constant k using first-order kinetic equation. Results indicated that significant reduction in stability and antibacterial activity of azithromycin solution when subjected to 70 and 100°C for 24 h. While stability of tetracycline was significantly reduced when subjected to 70 and 100°C for 24 h. However no significant reduction in antibacterial activity of tetracycline was observed at respective temperatures when compared with that of control. In addition, the stabilities of azithromycin and tetracycline were found to be decreased in acidic pH 4-5. The results of the present study revealed the high risk of contamination of milk sample with veterinary antibiotics and also demonstrated the effect of temperature and pH on stability of antibiotics. Therefore the study suggest that the qualitative and quantitative screening of milk for the presence of antibiotics need to be strictly performed to ensure safe drinking milk for consumers.
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Project description:Riemerella anatipestifer is an important pathogen in waterfowl, and is generally multidrug resistant. This study assessed the current status of Riemerella anatipestifer antibiotic resistance and antibiotic-resistance genes (ARGs), compared the results of different detection methods, and evaluated a new method of studying the association between antibiotic resistance and ARGs in Riemerella anatipestifer. In this study, 51 strains of Riemerella anatipestifer were isolated from ducks on several farms, their resistance to 28 antibiotics was assessed, and the isolates were subjected to whole-genome sequencing. The number of ARGs carried by Riemerella anatipestifer was predicted, compared, and analyzed, and the consistency between ARGs and antibiotic-resistance phenotypes was assessed. The potential for loss of resistance genes during the sequencing and assembly of genome-wide framework map was assessed, and a new ARG detection method was pilot tested. The 51 strains of Riemerella anatipestifer were multidrug resistant (MDR) and had high level of resistance to aminoglycosides, trimethoprim, lincosamides, polypeptides, and macrolides. Based on the genome-wide framework map of the 51 strains, 3 local databases of ABRicate software and 1 online database of CARD website were used to detect ARGs, and a mean of 4 to 5 ARGs were identified per isolate. Although the detection results differed according to the database used, the general performance was consistent. The online website detected more types of ARGs than the ABRicate software. The association between ARGs and antibiotic-resistance phenotypes was assessed, and the ermF gene was identified as a possible key ARGs regulating macrolide resistance of Riemerella anatipestifer. The method used to investigate and detect Riemerella anatipestifer ARGs was convenient and rapid, and had strong accuracy and pertinence. The ARGs detection method reported here combined the advantages of PCR and genome detection, and could greatly reduce workload and detect ARGs more precisely.
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Project description:Salmonella detection in equids at slaughter
| PRJEB61681 | ENA
Project description:ObjectiveSalmonella spp. are one of the leading foodborne pathogens worldwide naturally found in the intestines of many animals. People that are in direct contact with the infected animals or their cages may become ill. The aim of this study was to determine the prevalence, antibiogram and virulence genes associated with Salmonella serovars from fecal samples of animals intended for consumption in Southern Benin.ResultsOut of a total of 406 samples, 2.46% were positive. The isolates identified were multidrug-resistant Salmonella spp. to penicillins, first generation cephalosporins and some aminoglycosides. All Salmonella isolates produced invA gene of 284 bp, fimA of 85 bp and stn of 260 bp. The spvC gene (571 bp) was present in 10% of the isolates whereas the spvR gene (310 bp) was found in 20% of the isolates. The control strain possessed all the tested genes. The invA gene implies that strains are able to invade epithelial cells. The fimA and stn genes present in all isolates show that they are capable of causing gastrointestinal illness in humans. The presence of spvC and spvR genes suggests the possibility of these strains to produce toxins.
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Project description:Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time- and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.
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Project description:BackgroundQuantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods.ResultsDNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/?L. Forty-seven extracts had concentrations greater than 17.5 ng/?L, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ ?L), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/?L to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/?L and 0.25 ng/?L respectively. The qPCR LOQ varied by cell type (5.72?×?10-3 ng/?L for E. coli, 2.66?×?10-3 ng/?L, for B. cereus, 3.78?×?10-3 ng/?L for B. thailandensis, and 7.67?×?10-4 ng/?L for S. cerevisiae). A number of samples were below the UV spectroscopy (n?=?27), flurometry (n?=?15), and qPCR (n?=?3) LOQ.ConclusionThe presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.
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