An analysis of locally purchased imported and domestic foods' antibiotic resistance traits: verification of present plasmids, identification of common genes, and potential public health concerns

Call, Douglas

Davis, Margaret

Orfe, Lisa

Tokuno, Deven

Washington State University  institutional repository

Imported food can serve as a fomite for antibiotic resistance genesDeven Tokuno1, Lisa H. Orfe1, Margaret A. Davis1,2, and Douglas R. Call1,21Department of Veterinary Microbiology and Pathology, and 2Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WADiscussionWe selected uncooked farmed shrimp from Thailand and Indonesia and fresh wild shrimp from Mexico and the United States for these studies.  We extracted bulk DNA from these products and introduced the DNA to a lab strain of E. coli by electroporation followed by selection on antibiotic-containing agar plates.  This procedure should permit us to recover antibiotic resistance gene-bearing plasmids assuming that they can be maintained by E. coli.   Presence of recovered plasmids is being verified using plasmid profiling followed by genotyping using an antimicrobial resistance genotyping microarray (analysis in process). There is a slight tendency for greater resistance for farmed shrimp compared to wild shrimp (avg. 6.5 vs. 5.5 phenotypes), which confirms our hypothesis. Nevertheless, conclusions about differences between farmed and wild shrimp must be tempered by potential confounding factors including: (1) the samples originated from different geographic locations; (2) contamination could have arisen during packaging or repackaging; (3) freezing may bias recovery; (4) different shrimp species are included in our analysis; and (5) we have limited statistical power for comparisons. Table 1: Shrimp location and purchasing informationIntroductionAntibiotic use in food animal production is considered a significant factor in the emergence, amplification, and dissemination of antibiotic resistant bacteria. Commensurate public health concerns could lead to policies that significantly curtail antibiotic use in U.S. food animal production. While such policies may be justified, we are concerned that it would not necessarily result in the expected public health benefits. One counter pressure to such policies is the use of antibiotics outside of U.S. borders where regulation may be negligible. Importation of food products from these regions could therefore introduce antibiotic resistance elements into the United States. As an initial examination of this idea we chose to determine if antibiotic resistance genes can be readily recovered from imported farmed and fresh shrimp. AcknowledgementsWe are grateful for assistance from other members of the Call lab as well as for the financial assistance from the CAHNRS Undergraduate Scholar Fellowship program. This work was supported in part by the Washington State Agricultural Research Center, and from the Agricultural Animal Health Program, Washington State University College of Veterinary Medicine.Next Steps• Further analysis of microarray results• In cases where there is a phenotype but no genotype (Table 4), we will use expression libraries to identify unknown/novel genes.• The genotype date will also provide genetic markers that we can use to quantify the relative “load” of resistance genes in different shrimp samples.Hypothesis: Antibiotic resistance genes will be recovered from farmed shrimp products at a higher frequency compared with wild-caught shrimp products.Objective 1: Determine if antibiotic resistance traits can be recovered from uncooked shrimp products purchased from local markets.Objective 2: Characterize antibiotic resistance traits (multidrug resistance profiles, genotypes, plasmid mobility) and determine the prevalence of resistance traits in food products.Results• To date we have extracted and electroporated DNA from 100 samples and we have recovered 76 E. coli that are resistant to at least one antibiotic. • Several of these E. coli express multidrug resistance traits• Thus far, microarray results have indicated that multiple antibiotic resistance genes are present regardless of extraction method and sample origin. The rinsate procedure tended to provide a better yield of resistant E. coli .• Assessment of the diversity of resistance genotypes using the microarray indicates variation, though analysis is still in process.• Our findings support our hypothesis that locally purchased imported food products can be contaminated with bacteria that harbor antibiotic resistance traits.Why Shrimp?The United States is the largest importer of shrimp in the world and shrimp are the top-selling seafood in the United States. In total the industry is worth approximately 60 billion U.S. dollars globally with 99% of farmed shrimp being produced in developing countries in tropical regions such as Southeast Asia. This effort includes some 110,000 warm-water shrimp farms, covering around 3.2 million acres, and the black tiger shrimp (Penaeus monodon) is the most commonly cultured species. Disease challenges plague the industry, especially Vibrio species, some of which are zoonotic. Antibiotics are used routinely, including fluoroquinolones (norfloxacin and enrofloxacin), flurazolidone, oxolinic acid, chloramphenicol, oxytetracycline, trimethoprim, sulphadiazine, and  streptomycin. One study estimated that in 1994 Thailand alone used 500-600 tonnes of antibiotics for shrimp production. Materials and Methods• We tested ten antibiotics using agar break-point tests (Table 1).• Samples: from imported and domestic fresh and farmed shrimp (Table 2).• We collected shrimp rinsate by shaking 1g of shrimp material in 1 mL ATL buffer (Qiagen) overnight. Alternatively, 1 g of shrimp tissue was “stomached” in 10 ml PBS. • DNA was extracted from sample material using a Qiagen Dneasy kit as per manufacturers protocols. • Extracted DNA was used to transform electrocompetent S17-λpirEscherichia coli that were subsequently selected on various antibiotic plates. Note: this procedure will only capture antibiotic resistance genes that are harbored on plasmids that can be maintained and expressed by E. coli.• Each location’s shrimp underwent six separate trials, isolated from independent electroporation experiments. • Resultant transformants were tested for multi-drug resistance and will undergo plasmid profiling (Kado et al, 1991).• Genes responsible for conveying antibiotic resistance will be identified using an antibiotic resistance microarray (Davis et al, 2010).• Results from the microarray will be verified using PCR and gene specific primers.Figure 1:Microarray slideshows genotypes, where each “spot” represents positive hybridization between sample DNA and a resistance gene probe.A) R=Rinsate and S=Stomached.  B)Amp = ampicillin; Km = Kanamycin; Tet = tetracycline; Gm = Gentamicin; S = Streptomycin; Cef = Cefoxitin G = Sulfadiazine.Antibiotic resistance: shrimp isolates demonstrated resistance when  E. Coli transformants grew on breakpoint antibiotic plates (as defined in Table 2).Table 2: antibiotic classes and antibiotics used Antibiotic concentrations were used as per recommended break point data, depending on the drug (except for Ampicillin, normal breakpoint 32µg/ml.Country of OriginFresh/FrozenRaw/ CookedPackaging LocationBrand Farmed/WildThailand Frozen Raw Pleasonton, CAWaterfront Bistro (Safeway, Pullman, WA) FarmedIndonesia Frozen Raw Pleasonton, CAWaterfront Bistro (Safeway, Pullman, WA) FarmedMexicoFresh frozenRaw Gulf of MexicoMeat and Seafood Dept. (Safeway, Pullman, WA) WildUnited StatesFresh frozenRaw Gulf of MexicoMeat and Seafood Dept.(Rosauer’s, Moscow, ID) WildLocation R or SA Ab Res ProfileB Resistance GenotypeIndonesiaR AmpKmTetGmS bla-TEM1; aph(3’)-IIaS AmpGTetGmSCef tetA; aadA1ThailandR TetGmS aadA1; aadA2; tetAS TetGmS addA1; aadA2; tetA; dfrA1United StatesR AmpKmTetGmS bla-TEM1; aph(3')-IIaS KmTetGmSG bla-TEM1; aph(3’)-IIaTable 4: Microarray resultsTable 3:  Single versus multiple drug resistanceLocation % Single Resistance (1-2 antibiotics) Multiple Resistance (greater than 3 antibiotic Classes)Indonesia 29.4% 70.6%Thailand 11% 89%Mexico 15% 85%United States 42.1% 57.9%Aminoglycosides Amikacin (30 µg/ml); Gentamicin (16 µg/ml); Kanamycin (50 µg/ml);Streptomycin (10 µg/ml)Cephalosporins Cefoxitin (2nd generation) (8 µg/ml) Macrolides Erythromycin (8 µg/ml)Beta-lactams Ampicillin (100 µg/ml)Quinolones Nalidixic acid (30 µg/ml)Sulfonamides Sulfadiazine (76 µg/ml); Sulfamethoxazole (512 µg/ml)Tetracyclines Tetracycline (50 µg/ml)Phenicols Florfenicol (50 µg/mL)While most of the antibiotic resistances are genetically explained, some (notably the Indonesia stomached sample which is resistant to beta-lactams with no detected beta-lactamase) are lacking a detected resistance gene (phenotype in bold).

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Imported food can serve as a fomite for antibiotic resistance genes

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Imported food can serve as a fomite for antibiotic resistance genes

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