Location: Meat Safety and Quality
2019 Annual Report
Objectives
Objective 1. Molecular characterization including whole genome sequencing and transcriptomic characterization of foodborne bacteria, including pathogens and commensals, exposed to various physiologically relevant conditions reflective of the production continuum.
Sub-objective 1.A: De novo, whole genome sequencing and metagenomic profiling of the microbial community present in bovine rectoanal mucosa (RAM) swab samples.
Sub-objective 1.B: Characterize the genomic, phenotypic and transcriptional differences present in clinically important STEC and Salmonella serotypes exposed to different physiological relevant conditions in order to identify virulence and regulatory control mechanisms.
Objective 2. Characterize the ecological niches and reservoirs to identify mechanisms of foodborne pathogen, especially biofilms, for their ability to colonize and persist leading to the development of intervention strategies.
Sub-objective 2.A: Molecular mechanisms of biofilm formation.
Sub-objective 2.B: Association between biofilm formation, antibiotic resistance, and sanitizer tolerance.
Objective 3. Development and validation of various antimicrobial resistance detection methodologies including culture and genomic techniques, such as whole genome sequencing.
Sub-objective 3.A: Evaluation of culture based methods for the detection of bacteria resistant to antimicrobials important to human medicine.
Sub-objective 3.B: Development of genomic methods for the detection of antimicrobial resistance elements.
Approach
The cost of food borne illness and the loss of productivity in the United States is reported to be greater than $14 billion a year. While research efforts have resulted in great strides in tracking contamination entry points and identifying mitigation strategies, outlier events continue to occur and complete prevention of foodborne pathogens entering the food chain remains an elusive goal. Attaining this goal is challenging in part because many of the target pathogens live in dynamic and complicated communities, likely not even causing disease in their host reservoir. In addition, a better understanding of the use of antimicrobial agents in animal production and the possible impact on foodborne pathogens acquiring resistance has become a top priority for many government agencies and health care advocates. The project described here will provide new information about these issues by helping to better understand the different colonization sites and how various pathogens survive and interact with their respective bacterial communities. Further, we will characterize population differences within these foodborne bacteria, focusing on those that enhance an organism’s ability to cause human illness. Ultimately, the overall aim of this project is to provide new information about pathogen (predominantly Shiga toxin-containing Escherichia coli (STEC) and Salmonella enterica) persistence and survival in a variety of environments that position them for entry into the food supply.
Progress Report
Under Objective 1, we have made substantial progress in comparative transcription of Shiga toxin-containing Escherichia coli clinical and environmental strains. Previous experiments to phenotype strains using Phenotyping Microarrays demonstrated a strong correlation between metabolic rates in STEC O157 that associated with strains being from an environmental or clinical source. To better understand the described phenotypes, comparison of mRNA transcription was completed to determine the correlation between metabolic phenotype and mRNA abundance for the environmental and clinical strains. Overall, there were 564 genes with 3 times more mRNA abundance in one strain verse the other. The majority of the transcriptional differences were from the clinical strain. These involved carbon and amino acid substrates that correlated with the metabolic phenotyping. The differentially expressed genes in the clinical strain are involved in utilization of different carbohydrates associated with plants and degradation of amino acids, specifically lysine, threonine and glutamic acid. Also, the virulence genes, intimin, espD, espA and Shiga toxin had greater expression in the clinical strain versus the environmental strain. The environmental strains showed an increased ability to utilize n-acetyl-D-Galactosamine compared to the clinical strain but there was no difference by mRNA transcription between the two strains. Work is ongoing to identify the transcription regulators that are responsible for the differences in transcription and how they influence the observed phenotypes.
We also collected fecal swab samples from 180 calves that were either mass-medicated with a macrolide antibiotic or not (90 mass-medicated, 90 control). Swab samples were collected pre mass-medication, day 0, and post mass-medication on days 1, 2, 9 and 28 of the study. Metagenomic DNA is being isolated from these samples which will then be sequenced using a long-read sequencing platform. Changes in microbial community structure over the course of the study in both mass-medicated and control populations will be evaluated using the data analysis pipeline WIMP (“What’s In My Pot”). qPCR assays for bacterial 16s RNA and eukaryotic 18s RNA also will be used to assess relative abundance of host (cattle) and bacterial DNA template within the metagenomic DNA samples evaluated.
Under Objective 2, we continue to work with the meat industry towards understanding the impact of biofilm formation on meat safety at commercial plants, and also evaluate STEC O157:H7 tolerance against common sanitizers in order to help the industry identify the most effective product as well as develop practical and cost-effective sanitization protocols. To overcome the challenge posed by the strong biofilm forming STEC strains with a variety of complex tolerance mechanisms, a multi-faceted approach using multiple sanitizers or combining sanitizer usage with other cleaning methods has been shown to enhance effectiveness due to the synergistic effect. In addition, since the meat processing equipment with scratches and other hard-to access areas such as the underside of conveyor belts would serve as viable niches for biofilm development, the meat industry is in need of not only effective sanitizer products but also an easy-to-implement protocol that can be applied to the processing environment and the various equipment including those hard-to-reach areas. As requested by the major beef processors and the sanitizer producers, we evaluated the effectiveness of novel sanitizer products that use a combination of various chemical reagents that can be deployed as foam, liquid solution or fog to cover the different processing areas as needed. Results indicate that the Decon-7TM sanitizer, a product consisting of quaternary ammonium compound, hydrogen peroxide, and the accelerator diacetin, was effective even at 10% of its recommended concentration in reducing STEC O157:H7 and Salmonella biofilms on stainless steel and polyvinyl chloride plastic surfaces to a non-detectable level, and it also prevented pathogen post-sanitization prevalence. Our mechanism study using scanning electron microscope further revealed that this product was able to weaken and dissolve bacterial extracellular polymeric substance that facilitates the connection between bacterial cells and the contact surface. The biofilm architecture became less intact while treated by increasing concentrations of Decon-7TM foam and the bacteria appeared to undergo certain morphology alternation with shortened cell length and compromised membrane integrity, indicating biofilm mass removal and loss of bacterial viability. Therefore, this product with an easy-to-implement protocol covering all areas in a processing environment provides an option that the meat industry may consider in the biofilm prevention and control program as it is effective in biofilm inactivation and prevalence prevention.
In addition, the effect of residual amounts of common sanitizers on bacterial biofilm formation and sanitizer susceptibility was investigated. Multiple generations of bacterial growth with and without the residual amounts of sanitizers were tested and compared. Sanitizer sub-lethal concentration range that was able to induce survival stress and reduce replication rate has been identified and is being used to repeat the phenotype screening test. The selected isolates with repeated exposure to the sub-lethal concentrations of sanitizers will be analyzed with whole genome sequencing to identify potential genomic alteration by the treatment.
Under Objective 3, to address concerns regarding impact of antimicrobial use on the occurrence of antimicrobial resistance (AMR) comprising both antimicrobial resistant bacteria (ARB) and antimicrobial resistance genes (ARGs), we have completed projects to determine the impact of tylosin inclusion in feed to prevent liver abscesses on levels of ARB, compared the levels of ARB in colon contents, on carcasses, and in trim of cull cattle produced with and without the use of antibiotics and compared the levels of ARB in retail ground beef products with and without “raised without antibiotics” claims obtained from 6 U.S. cities. In the first project, a total of 1,920 samples were cultured to determine the impact of tylosin inclusion in feed to prevent liver abscess on levels of 5 ARB in cattle production and processing environments. Tylosin in feed had no impact on detection of ARB. These results suggest that this application of tylosin has no or minimal impact on antimicrobial resistance in cattle production and processing environments. In the second project, a total of 1,129 samples were cultured to compare the levels of 5 ARB in colon contents, on carcasses, and in trim of cull cattle produced with and without the use of antibiotics. Additionally, for 535 colon content samples qPCR was performed to compare the levels of 10 ARGs. Generally similar AMR levels were observed regardless of antibiotic use. These results indicate that cull cow production has a minimal impact on AMR in feces and no impact on AMR presence on carcasses and in trimmings. In the third project, a total of 599 samples were cultured to compare the levels of 5 ARB in retail ground beef products with and without “raised without antibiotics” claims obtained from 6 U.S. cities. Additionally, qPCRs to determine the levels of 10 ARGs were performed for all ground beef samples. AMR levels were generally similar between products with and without “raised without antibiotics” claims. These results strengthen prior studies demonstrating that antibiotic uses during beef cattle production minimally impact AMR occurrence and that processing interventions effectively remove AMR.
Also to better understand AMR at the livestock-wildlife interface, a joint collaboration between ARS, the National Wildlife Research Center, the University of Wyoming, and McGill University investigated the genetic context of AMR Escherichia coli within beef cattle production systems in Northern Colorado. These analyses specifically targeted E. coli isolates with high-priority AMR (resistance to cephalosporins and ciprofloxacin) that were collected from the feces of cattle, raccoons, and mice. In total, more than 1,000 fecal samples were analyzed and screened for AMR E. coli. Following extensive phenotypic testing, a subset of 40 E. coli isolates were selected for comprehensive genetic analysis on the basis of their presence in cattle and wildlife, distribution at different livestock facilities, and composite susceptibilities to 18 different antibiotics. This study showed that cattle and wildlife harbored highly similar AMR E. coli, and that nearly identical isolates were present at multiple farms within livestock and wildlife hosts. In total, 72 chromosomal AMR determinants were detected, with 45 of these found in all isolates tested. The genetic conservation observed between AMR E. coli isolates from cattle and wildlife suggest a complex AMR livestock ecology that has inputs from multiple sources.
Accomplishments
1. Cropland amendment with beef cattle manure minimally impacts antimicrobial resistance. Concerns have been raised that using beef cattle manure to fertilize croplands increases the amount of antimicrobial resistant bacteria in these soils. If the increases persist until crops are planted, this could increase food-animal and human exposures to antimicrobial resistant bacteria through feed and produce. ARS scientists in Clay Center, Nebraska, and colleagues treated one farm each in Nebraska, North Dakota, and South Dakota with: beef cattle manure, inorganic fertilizer, or no fertilizer as a control.
Results indicate manure amendment did not change the levels of 8 antimicrobial resistance bacteria measured. Manure amendment did not increase antimicrobial resistance levels for 8 of the 10 antimicrobial resistance genes measured. For the other two antimicrobial resistance genes, AMR increases in manure amendment croplands only occurred at one location, were transient, and generally were within the normal variation observed for control croplands. Thus, we conclude that the common practice of land applying beef cattle manure in the Upper Midwest region of the U.S. likely has extremely minimal impact on environmental antimicrobial resistance levels, feed safety, food safety, animal health, and human health.
Review Publications
Rotundo, L., Boccia, F., Fratamico, P.M., Xu, A., Sommers, C.H., Liu, Y., Bono, J.L., Pepe, T. 2018. Draft genome sequences of seven strains of Shiga toxin-producing Escherichia coli O111 with variation in their sensitivity to novobiocin. Microbiology Resource Announcements. 7(10). https://doi.org/10.1128/MRA.01030-18.
Harhay, D.M., Smith, T.P.L., Harhay, G.P., Loneragan, G.H., Webb, H.E., Bugarel, M., Haley, B.J., Kim, S.W., Van Kessel, J.S. 2018. Complete closed genome sequences of three Salmonella enterica subsp. enterica Serovar Dublin strains isolated from cattle at harvest. Microbiology Resource Announcements. 7:e01334-18. https://doi.org/10.1128/MRA.01334-18.
Haley, B.J., Smith, T.P., Harhay, G.P., Loneragan, G.H., Webb, H.E., Bugarel, M., Kim, S., Van Kessel, J.S., Harhay, D.M. 2019. Complete genome sequence of a Salmonella enterica subsp. enterica serovar Fresno isolate recovered from beef cattle lymph nodes. Microbiology Resource Announcements. 8(2):e01338-18. https://doi.org/10.1128/MRA.01338-18.
Franz, E., Rotariu, O., Lopes, B.S., MacRae, M., Bono, J.L., Laing, C., Gannon, V., Söderlund, R., van Hoek, A., Friesema, I., French, N.P., George, T., Biggs, P.J., Jaros, P., Rivas, M., Chinen, I., Campos, J., Jernberg, C., Gobius, K., Mellor, G.E., Chandry, P., Perez-Reche, F., Forbes, K.J., Strachan, N. 2019. Phylogeographic analysis reveals multiple international transmission events have driven the global emergence of Escherichia coli O157:H7. Clinical Infectious Diseases. 69(3):428-437. https://doi.org/10.1093/cid/ciy919.
Bugarel, M., Cook, P.W., Den Bakker, H.C., Harhay, D.M., Nightingale, K.K., Loneragan, G.H. 2019. Complete genome sequences of four Salmonella enterica strains (including those of serotypes Montevideo, Mbandaka, and Lubbock) isolated from peripheral lymph nodes of healthy cattle. Microbiology Resource Announcements. 8(2):e01450-18. https://doi.org/10.1128/MRA.01450-18.
