Objectives. Antibiotics uses in food animals 3/25/2018. California Dairy Productions. Antimicrobial Resistance in the Animal Production Environment

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1 Antimicrobial Resistance in the Animal Production Environment Xunde Li Western Institute for Food Safety and Security Department of Population Health and Reproduction University of California Davis Objectives Describe the impact of animal agriculture on groundwater with a focus on antimicrobial resistance. Identify antimicrobial resistance patterns and risk factors on dairy farms. Recognize the significance of managements for reducing antimicrobial resistance in farm environment. 1 2 Problems of antibiotic uses Antibiotics uses in food animals 80% of are used in farm animals 17 classes 41 active ingredients in that are approved as AMDs for use in foodproducing animals in the U.S. (FDA 2012 Annual summary Report) Antibiotic Resistance is a food safety problem from the Farm to the Table om-farm-to-table.html California Dairy Productions 5 6 1

2 Concentrated Animal Feeding Operations (CAFOs) are considered a key biological and environmental reservoirs of AMR genes that impact farm-to-fork food safety and public health (Gilchrist et al., 2007; Marshall and Levy, 2011) Concentration of E. coli in different areas of two dairy farms in northern California Dairy production area E. coli concentration Calf hutch water (cfu/100 ml) soil (cfu/100 g) Cow pen soil (cfu/100 g) Flush/lagoon slurry (cfu/100 ml) Slurry irrigated fields (cfu/100 g) Groundwater (cfu/100ml) Groundwater under the influence of these high surface microbial loadings had substantially fewer bacteria (3- to 7-log10 reduction) compare to surface samples Dairy farm Antibiotic resistant E. coli on two dairy farms in northern California Calf hutches No. resistant /no. tested Heifer/ cow pen solids Flush slurry or lagoon Irrigated field Ground water I 3/4 5/15 2/11 3/4 1/1 II 3/5 6/18 7/12 4/7 0/3 Total 6/9 (67%) 11/33 (33%) 9/23 (39%) 7/11 (64%) 1/4 (25%) E. coli was mainly resistant to tetracycline (25.0 %), cefoxitin (25.0 %), amoxicillin/clavulanic acid (23.8 %), and ampicillin (22.5 %) % of all were found to be multiantibiotic resistant (resistant to 3 ). The isolate from groundwater showed similar resistance patterns as from surface. 9 Negative binomial regression model of the association between source of sample and season of sampling on the risk of E. coli being resistant to an increasing number of Risk factor Sample source Unadjusted no. of E. coli is resistant to a Calf hutches (water or soil) 3.3 Coefficient P value 95 % CI Heifer/cow (pen solid) ( 2.1, 0.3) Flush lane or lagoon (slurry) <0.001 ( 3.3, 1.5) Irrigated field (soil) ( 1.6, 0.02) Groundwater well (water) ( 5.3, 1.7) Season Feb. 2.2 April ( 2.4, 0.2) Sept. Oct <0.001 ( 1.1, 0.5) a Crude or unadjusted mean number of that E. coli strains were resistant to (i.e., source and season not adjusted for each the other) (per gram solids or 100 ml liquids) 10 Monitoring microbial pathogens and antimicrobial resistance in the Central Valley Black: Dairy farms Red: Studied dairy farms (8) White: Survey domestic (200) 11 Survey of indicator and pathogenic bacteria in groundwater from domestic with likely dairy influence, in the Central Valley, California ( ). Concentration Well types Bacteria (CFU/100ml) positive Mean (±SD) Near-dairy Wells (with dairy influence) (N=132) within dairy facility (N=12) outside of dairy facilities (N=120) Generic E. coli 1 (8.3) 0.72 Enterococcus spp. 3 (25.0) 0.13 (0.15) Campylobacter spp. 0 (0) NA Salmonella spp. 0 (0) NA E. coli O157:H7 0 (0) NA Generic E. coli 5 (4.2) 0.26 (0.39) Enterococcus spp. 7 (5.8) (43.33) Campylobacter spp. 0 (0) NA Salmonella spp. 0 (0) NA E. coli O157:H7 0 (0) NA 12 2

3 E. coli Enterococcus 3/25/2018 Survey of indicator and pathogenic bacteria in groundwater from domestic without likely dairy influence, in the Central Valley, California ( ). Well types Bacteria positive Non-Dairy Wells (without dairy influence) (N=68) Concentration (CFU/100ml) Mean (±SD) Generic E. coli 4 (5.9) 1.93 (3.33) Enterococcus spp. 7 (10.3) (37.15) Campylobacter spp. 0 (0) NA Salmonella spp. 0 (0) NA E. coli O157:H7 0 (0) NA 13 Factors associated to the concentrations of Enterococcus in groundwater from domestic in the Central Valley, California ( ). Factor Coef. Std. Err. P value 95% CI Well designation Non-Dairy well Near-Dairy well (0.408, 3.393) Potassium ( , ) Season Spring Summer ( , 7.552) Fall (0.251, 3.593) Winter (-1.077, 3.010) Referent category for categorical variable Journal of Environmental Quality, 2015; 44(5): Number of antibiotic resistant of generic E. coli and Enterococcus isolated from groundwater Type of Bacteria Campaign No. tested No. resistant to 1 No. resistant to Drugs most likely 3 resistant to On-dairy Monitoring NA NA Azithromycin; chloramphenicol; Near-dairy trimethoprim/sulfam ethoxazole; Non-dairy tetracycline. Total (100%) 7 (63.6%) On-dairy Monitoring Tigecycline; quinupristin/dalfopri stin; linezolid, Near-dairy chloramphenicol; erythromycin; Non-dairy iprofloxacin; tetracycline Total (100%) 31 (86.1%) 15 Dairy account for ~ 8% of U.S. domestic beef production 25% of U.S. nonfed beef available for consumption in the U.S. 18% of U.S. ground beef 16 Survey of antimicrobial resistance in cull dairy : a two-year study 6 dairies in the Central Valley near VMTRC Year1: ; ~ 250 cattle sampled Year 2: ; 460 cattle sampled Seasonal sampling Phenotypic resistance Collect data of risk factors

4 Antimicrobial resistance of generic E. coli and Enterococcus from cull dairy cattle in California ( , ). Social network analysis of risk factors contributing to antibiotic resistance Bacteria E. coli Enterococcus Salmonella No. tested Yr 1 (269) Yr 2 (322) Yr 1 (264) Yr 2 (292) Yr 1 (8) Yr 2 (63) No. (%) resistant to 1 antibiotic 62 (23%) 88 (27%) 213 (81%) 256 (87%) 3 (38%) 25 (40%) No. resistant to 3 11 (4%) 14 (4%) 117 (44%) 90 (30%) 2 (25%) 6 (10%) Most common drug resistance Tetracycline (22%) Tetracycline (21%) Lincomycin (73%) Lincomycin (76%) Ampicillin (38%) Tetracycline (29%) Drug management o Record o Training Drug use practice o Intervals o Regular patterns Hygiene etc Tracks of dairy productions Antibiotic resistance of generic E. coli in different dairy production and management stages Hutch calves Midlate Fresh Fresh Midlact Preg late lact Post wean Breeding Springer / close up Hospital Hospital Close up Far-off 21 Resistant to >1 drug Resistant > 3 drugs 22 Overall antimicrobial resistance of E. coli from cattle in different management units in a large dairy farm in central California, 2016 (59, with each isolate from individual animals) Class of Antimicrobials Aminoglycosides Cephalosporin Drugs susceptible (S) intermediate resistant (IR) resistant (R) Gentamycin 56 (94.9) 1 (1.7) 2 (3.4) Streptomycin 53 (89.8) 0 (0) 6 (10.2) Cefoxitin 57 (96.6) 0 (0) 2 (3.4) Ceftiofur 56 ( (1.7) 2 (3.4) Ceftriaxone 55 (93.2) 1 (1.7) 3 (5.1) Chloramphenicol Chloramphenicol 52 (88.1) 2 (3.4) 5 (8.5) Macrolides Azithromycin 59 (100) 0 (0) 0 (0) Penicillin Amoxicillin/clavulanic acid 58 (98.3) 1 (1.7) 0 (0) Ampicillin 56 (94.9) 0 (0) 3 (5.1) Quinolones Ciprofloxacin 57 (96.6) 0 (0) 2 (3.4) Nalidixic acid 57 (96.6) 0 (0) 2 (3.4) Sulfonamides Sulfisoxazole 59 (100) 0 (0) 0 (0) Trimethoprim-sulfamethoxazole 51 (86.4) 0 (0) 8 (13.6) Tetracycline Tetracycline 46 (78.0) 0 (0) 13 (22.0) 23 Comparison of antibiotic resistance of generic E. coli and Salmonella Salmonella resistant Salmonella susceptible E. coli resistant E. coli susceptible 24 4

5 Antibiotic resistance of Enterococcus in different dairy production and management stages Resistant to >1 drug Resistant to >3 drug 25 Overall antimicrobial resistance of Enterococcus from cattle in different management units in a large dairy farm in central California, 2016 (59, with each isolate from individual animals). Class of Antimicrobials Drugs susceptible (S) intermediate resistant (IR) resistant (R) Gentamycin 44 (74.6) 0 (0) 15 (25.4) Aminoglycosides Kanamycin 22 (37.3) 0 (0) 37 (62.7) Streptomycin 52 (88.1) 0 (0) 7 (11.9) Chloramphenicol Chloramphenicol 39 (66.1) 9 (15.3) 11 (18.6) Glycopeptides Vancomycin 52 (88.1) 4 (6.8) 3 (5.1) Glycylcycline Tigecycline 47 (79.7) ND* ND* Lincosamides Lincomycin 10 (16.9) 4 (6.8) 45 (76.3) Lipopeptides Daptomycin 44 (74.6) ND* ND* Macrolides Erythromycin 21 (35.6) 21 (35.6) 17 (28.8) Tylosin tartrate 36 (61.0) 1 (1.7) 22 (37.3) Nitrofurans Nitrofurantoin 25 (42.4) 34 (57.6) 0 (0) Oxazolidinones Linezolid 20 (33.9) 10 (16.9) 29 (49.2) Penicillin Penicillin 57 (96.6) 0 (0) 2 (3.4) Quinolones Ciprofloxacin 6 (10.2) 14 (23.7) 39 (66.1) Streptogramins Quinupristin/dalfopristin 36 (61.0) 4 (6.8) 19 (32.2) Tetracycline Tetracycline 41 (69.5) 1 (1.7) 17 (28.8) *Not determined due to the MIC breakpoints outranged the CLSI criteria 26 1 Bacterial communities in dairy cattle at different management units BreedingHeifers CloseUpCows Fresh Cows FreshHeifers Far- Off Cows HutchCalves Hospital MidLactCows MidlateLactHeifers PregLateLactCows Post Wean Heifers Springer/ Close up 27 Veterinary Feed Directive General requirements Animal feed bearing or containing a VFD drug or a combination VFD drug (a VFD feed or combination VFD feed) may be fed to animals only by or upon a lawful VFD issued by a licensed veterinarian. VFD feed Animals a licensed veterinarian VFD 30 5

6 California Senate Bill No. 27 SB 27, Hill. Livestock: use of antimicrobial drugs. Approved by Governor, October 10, 2015 Implementation date 1/1/2018 Limit use of medically important antimicrobial drugs Beginning 1/1/2018, medically important AMDs shall not be administered to livestock unless ordered by a licensed veterinarian through a prescription or VFD, pursuant to a VCPR. (a) Medically important AMDs may be used when in the professional judgment of a licensed veterinarian. (b) Medically important AMDs may also be used when, in the professional judgment of a licensed veterinarian, it is needed for prophylaxis to address an elevated risk of contraction of a particular disease or infection. (c) Medically important AMDs shall not be administered to livestock solely for purposes of promoting weight gain or improving feed efficiency. (d) Medically important AMDs shall not be administered to livestock in a regular pattern unless the administration is consistent with subdivision (a). Medically important AMDs labeled by FDA for OTC sales can still be obtained from retailers with prescription or VFD from a licensed veterinarian Judicious use of Alternatives Management solutions Hygiene Animal husbandry Management Drug use practices Etc. Strategic use of alternatives to Vaccines Prebiotics Probiotics Immune modulators Etc Field sampling and filtering Antimicrobial susceptibility testing Minimum Inhibitory Concentration (MIC) method Clinical Laboratory Standard Institute (CLSI) criteria

7 Isolation of E. coli and Enterococcus 3/25/2018 Rapid diagnose Molecular approaches to characterize microbiome and resistome Genomic DNA extraction Sequencing library Sequencing, assembling construction and annotation GGAGAA PCR amplification of 16S rdna Sequencing and annotation Phylogenetic analysis of AMR genes Sample Genomic DNA Genomic DNA sheared DNA fragments ligated extraction into -2kb fragments into plasmid vector 37 Sequencing and annotation PCR amplification of functionallyselected genes Functional screen on antibiotic-containing media Transformation of electrocompetent E. coli 38 Summary What we know: Animal agriculture is considered one of the contributors to biological and environmental reservoirs of acquired resistance genes What don t know: The probability, the mode, the spatiotemporal trends of AMR in animal agriculture, and the correlations with antimicrobials use have not been well characterized What are the industries need: o Farm management strategies to optimize the use of antimicrobials while protecting animal health and welfare o Alternatives to traditional What we can do: o Enhance monitoring and surveillance o Basic and applied research o Collaborations and coordination amongst industries, academia, and government 39 Thank You 7

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