Progress 10/01/14 to 09/30/17
Outputs Target Audience:Data will be made available to dairy farmers, veterinarians, and NYS dairy industry professionals for the evaluation and implementation of management practices to reduce the dissemination of resistance to antimicrobials important in human medicine. Farm employees will be served by a decreased risk of acquiring fluoroquinolone-resistant infections on the farm, and consumers of milk and meat supplied by dairy farms will benefit from increased food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our lab technician has worked with our new postdoctoral associate to learn DNA extraction techniques and the postdoc has worked with the technician to learn the HGMF techniques used to test the antimicrobial susceptibility of the samples. The post doc also learned the shotgun sequencing protocol. How have the results been disseminated to communities of interest?Data analysis is still ongoing prior to dissemination of this information. We anticipate that it will be disseminated to scientists in the field through publications, and subsequently to our agricultural stakeholders through veterinarians. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts What was accomplished under these goals?
We prospectively monitored the effect of treating calves with enrofloxacin at approved label dosages on the selection and spread of resistance to fluoroquinolones using the enteric bacterium E. coli. We additionally identified fluoroquinolone resistance genes in these organisms, and sought to evaluate the disruption of the healthy fecal microbiota. To accomplish this, we isolated and characterized E. coli resistant to fluoroquinolone drugs and other common antimicrobials used in both human and veterinary medicine after treatment of calves with enrofloxacin or, as a control, tulathromycin, an antimicrobial of the macrolide class, comparing them to untreated calves. Importantly, these studies were conducted on a commercial dairy farm using production practices employed widely in the industry, and thus will likely produce broadly applicable results. Treatment of calves with enrofloxacine following the label indications (prevention of bovine respiratory disease, BRD) significantly increased the proportion of fecal E. coli resistant to the fluoroquinolones ciprofloxacin and enrofloxacin at 7 days (P-value < 0.0001) after initial administration when compared to control calves not receiving any antimicrobial treatment and calves treated with the macrolide drug tulahtromycin (following label instructions for this drug). The same trend was observed for nalidixic acid (P-value = 0.076), another fluoroquinolone drug. Calves in the enrofloxacine group had 3.8 (95% CI: 1.9-5.6) and 6.4 (95% CI: 4.5-8.4) odds for having a E. coli resistant to ciprofloxacin when compared to calves in the tulathromycin treatment group and the control group, respectively. This is a important finding because ciprofloxacin is a drug of crucial important to human medicine, and selection for resistant to this drug class shold be avoided when possible. The fact that preventive treatment of calves with a drug belonging to another class (macrolides) did not significantly affect shedding of ciprofloxacine resistant E. coli, indicates that maybe this could be a better drug to use to in calves at higher risk of developing pneunomia. After 28 and 56 days of administration of enrofloxacin, no significant difference in the proportion of E. coli resistant to these three antimicrobial drugs could be observed when compared to control and tulathromycin group. E. coli resistant to other drugs tested was not observed to be present at a propotion significantly different between treatment groups for differnet sampling days. This preliminary data supports the hypothesis that treatment of calves with enrofloxacin can significantly increase fecal shedding of E. coli resistant to the fluoroquinolone class. Current information with quantitative values for colony forming units per gram of feces (CFU/g) are current being analyzed, and will provide a more precise estimate of the effect of treatment with enrofloxacin on selection of E. coli resistant to ciprofloxacine at each sampling time point (7, 28 and 56 days after treatment). Furthermore, and evaluation of the impacts of treating calves with enrofloxacin and tulathromycin for prevention of BRD on enteric fecal microbiota at 7, 28 and 56 days after treatment.
Publications
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Progress 10/01/15 to 09/30/16
Outputs Target Audience:Data will be made available to dairy farmers, veterinarians, and NYS dairy industry professionals for the evaluation and implementation of management practices to reduce the dissemination of resistance to antimicrobials important in human medicine. Farm employees will be served by a decreased risk of acquiring fluoroquinolone-resistant infections on the farm, and consumers of milk and meat supplied by dairy farms will benefit from increased food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our lab technician has worked with our new postdoctoral associate to learn DNA extraction techniques and the postdoc has worked with the technician to learn the HGMF techniques used to test the antimicrobial susceptibility of the samples. The post doc also learned the shotgun sequencing protocol. How have the results been disseminated to communities of interest?Most of the year was dedicated to processing calf fecal samples, performing antimicrobial resistance testing, and performing the shotgun sequencing. Not enough data has been generated for a preliminary assessment of the results. What do you plan to do during the next reporting period to accomplish the goals?The remainder of the fecal samples will be tested for antimicrobial susceptibility. Repeated measures analysis of variance in SAS will be used to analyze the treatment group effects on E. coli and Salmonella over time. PCR will be used to identify the resistance genes in isolates resistant to ciprofloxacin. The shotgun metagenomic sequencing (WGS) will be used to compare the fecal microbiota from calves treated or not with enrofloxacin or tulathromycin.
Impacts What was accomplished under these goals?
Laboratory work is nearly complete on the isolation, characterization and quantification of resistant E. coli from the calf fecal samples. DNA extraction from theseE.coliisolates for further PCR and genotyping analysis has begun. Total DNA was extracted from a subset of 180 fecal samples. Shotgun metagenomics sequencing was performed and sequences were uploaded to the online metagenomics analysis server MG-RAST for phylogenetic and functional analysis. Some of these results are still pending.
Publications
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Progress 10/01/14 to 09/30/15
Outputs Target Audience:Data will be made available to dairy farmers, veterinarians, and NYS dairy industry professionals for the evaluation and implementation of management practices to reduce the dissemination of resistance to antimicrobials important in human medicine. Farm employees will be served by a decreased risk of acquiring fluoroquinolone-resistant infections on the farm, and consumers of milk and meat supplied by dairy farms will benefit from increased food safety. Changes/Problems:One change was made to the calf enrollment protocol. The first farm houses calves in both individual hutches outdoors and in individual pens in a barn. It was decided that more information could be obtained from enrolling and sampling more calves at one farm over a longer period of time, with both housing types represented, than from enrolling 10 calves per treatment group at 2 farms in succession. The numbers of completed sets of samples are as follows: control (n=21), enrofloxacin (n=22), and tulathromycin (n=24). Housing will be treated as a fixed effect in the analysis if it is significant and as a random effect if it is not significant. What opportunities for training and professional development has the project provided?A veterinary student from the Brazilian Mobility Program worked with us for 3 months enrolling calves and collecting and processing samples. Our lab technician has worked with our new postdoctoral associate to learn DNA extraction techniques and the postdoc has worked with the technician to learn the HGMF techniques used to test the antimicrobial susceptibility of the samples. How have the results been disseminated to communities of interest?Most of the year was dedicated to enrolling calves in the study and collecting samples. Not enough data has been generated for a preliminary assessment of the results. What do you plan to do during the next reporting period to accomplish the goals?The remainder of the fecal samples will be tested for antimicrobial susceptibility. A subset of isolates will be selected for characterization of phenotypic resistance using disk diffusion. From these, PCR will be used to identify the resistance genes in isolates resistant to ciprofloxacin. Shotgun metagenomic sequencing (WGS) will be used to characterize the fecal microbiota of 36 calves.
Impacts What was accomplished under these goals?
Complete sets of samples (enrollment day samples plus 8 subsequent samples) were collected from 67 calves this year. Samples were prepared and frozen for culture, enumeration and testing for antimicrobial resistance. All environmental samples were also collected and cultured. Laboratory work has begun on the isolation, characterization and quantification of resistant E. coli from these samples. DNA extraction has been started on the samples needed for the metagenomic analysis of fecal microbiota.
Publications
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