2012 Annual Report
1a.Objectives (from AD-416):
1. To elucidate and provide descriptive data, such as prevalence and/or trends, including antimicrobial susceptibilities, and molecular subtyping for foodborne pathogens in food animals through the animal sampling arm of the NARMs program.
The project will (1) continue to improve the standardization and quality control of methods used; and (2) where appropriate and necessary re-evaluate and/or develop improved sampling strategies to answer priority questions for NARMS in food animal production that are consistent with the overall NARMS goals and objectives as described on the NARMS web site.
2. Be a national resource of enteric bacterial isolates and resistance data for food animals from NARMS and US-VetNet. This resource will facilitate the identification and characterization of antimicrobial resistance as it emerges. Further, it will facilitate the identification and implementation of any new research needs by the complementary research project within this Unit. It is expected that the project will be highly responsive to requests for data from FDA-CVM, USDA-FSIS, and other stakeholders.
1b.Approach (from AD-416):
Continual comparison and evaluation of existing culture methodology and quality control of methods.
Where appropriate and necessary re-evaluate and/or develop improved sampling strategies to answer priority questions for NARMS in food animal production that are consistent with the overall NARMS goals and objectives as described on the NARMS web site.
In collaboration with a scientist in the Poultry Microbiology Safety Research Unit we studied the effect both broth and agar plating media exerted in the recovery of Salmonella species. We determined that different media and/or temperature combinations exert significant bias on the recovery of Salmonella serotypes. With collaborators in USDA-FSIS, from CRIS 6612-32000-006-06D (in Unit) and with USDA-APHIS scientists we compared the Unit’s polymerase chain reaction (PCR) based Salmonella serotyping with conventional antibody based serotyping and the molecular Luminex system and determined that the PCR was equal to conventional and Luminex methods; PCR based serotyping is now used routinely for our serotyping needs at a significant savings. Quality control procedures and antimicrobial susceptibility testing methods were reviewed monthly: three staff members were recertified during the annual proficiency testing for VetNet with scores exceeding 90%. We continued testing Salmonella, Campylobacter, generic E. coli and enterococci from USDA-FSIS regulatory programs for the National Antimicrobial Susceptibility Monitoring System (NARMS); all isolates were further subjected to pulsed field gel electrophoresis (PFGE) and all data were captured in the NARMS and USDA VetNet databases. A 2nd enzyme was used during PFGE for Salmonella to increase our level of confidence that isolates were related. NARMS and VetNet data were used to assist the USDA-FSIS and the Centers for Disease Control outbreaks investigations in food borne illnesses and by USDA-FSIS to use in their predictive analytics model and for their regulatory testing programs. Susceptibility data from on-farm sampling in the top three major swine producing states (Iowa, Minnesota, and North Carolina) plus Ohio was also included in NARMS. We participated in the USDA-APHIS National Animal Health Monitoring System Beef Cattle Feedlot study. Salmonella, Campylobacter, E. coli, enterococci and C. difficile isolates were isolated and are being characterized phenotypically and genotypically. The Unit culture collection now consists of over 119,000 well characterized isolates of Salmonella, Campylobacter, E. coli, Enterococcus, Listeria, Staphylococcus aureus, and Clostridium difficile, including control strains dating back to the early 1990’s. Isolates are maintained and used by scientists within and outside of our Unit. Isolates were furnished to collaborators by use of a material transfer agreement. These isolates are invaluable for present and future research needs to assess the emergence/development/transmission of antimicrobial resistance genes.
A food borne outbreak associated with Salmonella Heidelberg. Uncomplicated gastrointestinal illness from Salmonella infection usually resolves within five to seven days and is not treated. If treatment is needed, particularly in the young, elderly, and immunocompromised, antimicrobial resistant (AR) Salmonella may compromise treatment. To track the development of AR among Salmonella, isolates from food at retail, ill humans, and animals at slaughter were tested for resistance to a panel of antimicrobials as part of the National Antimicrobial Resistance Monitoring System (NARMS) by FDA, CDC and USDA-ARS scientists, respectively. Further characterization using pulsed gel electrophoresis (PFGE) assisted in determining if Salmonella strains are related to each other. In March 2011 a multi-state outbreak of Salmonella serotype Heidelberg infections in humans was closely monitored. CDC and FDA collaborators demonstrated that Salmonella Heidelberg isolates from ill humans closely matched Salmonella Heidelberg isolates from ground turkey product at retail stores. Antimicrobial resistance patterns also matched between these isolates. A recall of ground turkey was inititated as a result of these findings. ARS scientists involved with NARMS in Athens, GA, in collaboration with FSIS scientists, identified isolates from turkeys at slaughter that also closely matched the outbreak isolates. FDA, ARS and CDC, scientists also closely matched isolates originating from past routine NARMS surveillance of isolates from ground turkey at retail, turkey at slaughter and from ill human clinical isolates, respectively. This data is critical for public and veterinary health officials and regulatory agencies as they investigate food borne illness outbreaks. This data is also critical for scientists studying the development of antimicrobial resistance and in the development of mitigation strategies.
Prevalence of Salmonella in dairy cattle. Understanding the prevalence of Salmonella in animals on farms is necessary to control for bacteria entering the food supply. However, these studies are expensive and less costly testing for Salmonella is essential. Scientists in USDA-ARS (Athens, GA) and -APHIS conducted a national study on United States dairy farms to determine if the same information using pooled or composite fecal samples (less costly) for culture of Salmonella versus testing individual fecal samples (costly) could be obtained. The USDA’s National Animal Health Monitoring System Dairy 2007 study collected samples on dairy operations from 17 major dairy states. Among 116 operations sampled, 41.4% (n = 48) were positive by individual samples, 39.7% (n = 46) by pooled samples, and 49.1% (n = 57) by composite samples. Relative to individual samples to determine herd infection status, the sensitivity was 85.4% for composite fecal samples and 91.7% for pooled fecal samples. On 33.6% of operations (39/116), Salmonella was cultured from all 3 sample types (individual, pooled, and composite). Of these, 20 operations (51.3%) had exact Salmonella serotype matches in all 3 sample types. Composite fecal sampling is less costly and time consuming and provided similar results for detecting/identifying Salmonella in dairy herds. Therefore, it may be considered an alternative to culture of individual samples when assessing Salmonella status in dairy herds. These data are important for the dairy industry and other researchers as they develop and implement prevalence studies.
Multi-drug resistant NTS (non-Typhoidal Salmonella) is a major food safety concern worldwide. In collaboration with scientists at the Ohio State University ARS scientists in Athens, GA characterized multi-drug resistant (MDR) Salmonella serovar Havana, an emerging serovar reported from multiple sources including porcine, other food animals and the environment. Polymerase chain reaction and DNA sequencing were conducted to characterize resistance gene cassettes and class I integrons which are genes that are known to reside either within the bacteria’s chromosome or on a mobile genetic element, such as a plasmid, that can be transferred to other bacteria. An unusually large single integron was identified commonly among seven members of this serovar. This integron was found to carry gene cassettes that encode resistance to multiple classes of antimicrobials including sulphonamides, beta-lactamases, aminoglycosides and macrolides. The gene cassettes identified on the large 4kb integron include dfra16, blaPSE1, aadA2, and ereA resistance genes. Finding such a wide range of resistance against antimicrobial classes on a single integron is unique and implies the high propensity of this serovar to persist in the face of antimicrobial selective pressure and thus potentially become a major food safety concern. Additional genes that were not associated with the class I integrin included tetA(B), strA, strB and apha1-Iab. The findings underscore the significance of class 1 integrons in maintenance and persistence of MDR serovars of food safety significance and they are important in the development of mitigation strategies.
Prevalence of Salmonella in swine. Food borne illness is typically self-limiting and does not require antibiotics; however, when antibiotics are needed, resistant bacteria may be difficult to treat. To determine the prevalence Salmonella and characterize their susceptibility to antimicrobials USDA-ARS (Athens, GA) and -APHIS scientists cultured swine fecal samples as part of the National Animal Health Monitoring System’s Swine 2000 and 2006 studies in over 17 states. Overall, 6.2% of the samples and 34.2% of the farms were positive in 2000 while in 2006, 7.2% of the samples and 52.6% of the farms were positive. Salmonella serotypes Derby, Typhimurium var. 5- and Agona were the three top serotypes recovered in both study years. In both years Derby was most often resistant to streptomycin, sulfisoxazole and tetracycline. Most isolates were resistant to tetracycline, sulfisoxazole and streptomycin in both years. The percentage of isolates susceptible to all antimicrobials was 38.1% in 2000 and 20.4% in 2006 while the percentage resistant to three or more antimicrobials was 52.8% in 2000 and 57.7% in 2006. These data indicate that the prevalence of Salmonella remained relatively stable between years but the total number of positive farms increased. There was no change in the types of Salmonella recovered between study years and antimicrobial resistance was observed mostly in drugs that are not used to treat salmonellosis. These data are important to epidemiologists, veterinarians and public health officials as they monitor the health of food animals, status of the food supply, conduct food borne outbreak investigations, and develop mitigation strategies.
Tracking the emergence of antimicrobial resistant Salmonella Heidelberg. Surveillance conducted as part of the National Antimicrobial Resistance Monitoring System (NARMS) has shown a recent increase in extended-spectrum cephalosporin (ESC) resistance among Salmonella Heidelberg isolated from food animals at slaughter, retail meat, and ill humans. ARS scientists in Athens, GA and collaborators at the Centers for Disease Control and the Food and Drug Administration demonstrated that the 2009 increase in ESC resistance among Salmonella Heidelberg was caused mainly by the dissemination of blaCMY on 2 types of plasmids, IncI1 and IncA/C, in a variety of genetic backgrounds and is likely not the result of clonal expansion. This data is critical for public and veterinary health officials and regulatory agencies as they investigate food borne illness outbreaks. This data is also critical for scientists studying the development of antimicrobial resistance and in the development of mitigation strategies.
Haley, C.A., Dargatz, D.A., Bush, E.J., Erdman, M.M., Cray, P.J. 2012. Salmonella prevalence and antimicrobial susceptibility from the National Animal Health Monitoring System's (NAHMS) Swine 2000 and 2006 Studies. Journal of Food Protection. 75(3):428-436.
Karlsson, M.S., Joyce, K., Blickenstaff, K., Ball, T.A., Haro, J.H., Medalla, F., Cray, P.J., Zhao, S., Crump, J., Whichard, J. 2011. Antimicrobial susceptibility to azithromycin among Salmonella enterica isolated from the United States. Antimicrobial Agents and Chemotherapy. 55(9):3985-3989.
Blau, D.M., Mccluskey, B.J., Ladely, S.R., Dargatz, D.A., Cray, P.J., Ferris, K.E., Headrick, M.L. 2005. Salmonella on dairy operations in the United States: prevalence and antimicrobial drug susceptibility. Journal of Food Protection. 68(4):696-702.
Lindsey, R.L., Frye, J.G., Cray, P.J., Meinersmann, R.J. 2011. Microarray-based analysis of IncA/C Plasmid-Associated genes from multidrug-resistant Salmonella enterica. Applied and Environmental Microbiology. 77(19):6991-6999.
Folster, J.P., Pecic, G., Rickert, R., Taylor, J., Zhao, S., Cray, P.J., Whichard, J., Mcdermott, P. 2012. Characterization of multidrug-resistant Salmonella enterica serovar Heidelberg from a ground turkey-associated outbreak in the United States in 2011. Antimicrobial Agents and Chemotherapy. 56(6):3465-3466.
Cox Jr, N.A., Richardson, L.J., Cason Jr, J.A., Buhr, R.J., Vizzier-Thaxton, Y., Smith, D.P., Cray, P.J., Romanenghi, C.P., Pereira, L.B., Doyle, M.P. 2010. Comparison of neck skin excision and whole carcass rinse sampling methods for determining Salmonella prevalence and E. coli counts on broiler carcasses before and after immersion chilling. Journal of Food Protection. 73(5):976-980.
Guard, J.Y., Morales, C., Cray, P.J., Gast, R. K. 2011. Single nucleotide polymorphisms that differentiate two subpopulations of salmonella enteritidis within phage type. BMC Research Notes. 4:369
Kich, J.D., Coldebella, A., Mores, N., Nogueira, M.G., Cardoso, M., Fratamico, P.M., Call, J.E., Cray, P.J., Luchansky, J.B. 2011. Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil. International Journal of Food Microbiology. 151(3):307-313.