2013 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 completed studies on the effect of different broth and agar plating media as well as the impact of pH on the recovery of Salmonella serotypes from various sample types. Additional studies with other serotypes are planned. Lab personnel passed all proficiency testing and staff were recertified for PFGE. In collaboration with four University partners, the on-farm swine pilot was completed, data were summarized and presented to the FDA; each University partner was also given their state reports. Additional characterizations of isolates are being completed. In collaboration with a scientist in the Egg Quality and Safety Research Unit, we have begun sampling layer houses utilizing different caging systems. These houses were either newly constructed or renovated and were sampled prior to placement of birds; sampling has continued at measured intervals over time. The goal is to track both the emergence of Salmonella and Campylobacter within the birds and environment and more importantly the emergence and dissemination of antimicrobial resistance. Both the NAHMS Feedlot and NAHMS swine studies were completed in FY2012; however, we have continued to speciate and conduct antimicrobial susceptibility testing on Campylobacter isolates, speciate the enterococci isolates from the Swine study, conduct PFGE on the Salmonella isolates from the Swine study, and summarize the data. Collaborations are continuing with USDA-APHIS scientists in Fort Collins, CO and Ames, IA to complete the susceptibility testing and prepare manuscripts. Antimicrobial susceptibility testing on USDA-FSIS HACCP regulatory isolates for use in the NARMS program was conducted at the request of FSIS and FDA. Staff members also trained and transferred knowledge and know-how related to antimicrobial susceptibility testing to FSIS personnel; complete data sets were also transferred to FSIS. NARMS and VetNet data were used to assist the FSIS and the Centers for Disease Control foodborne outbreaks investigations and by FSIS to use in their predictive analytics model and for their regulatory testing programs. As a result of insufficient funds we did not accept data from VetNet collaborating centers, begin PFGE of methicillin staph aureus, or use a second enzyme on Campylobacter isolates. The Unit culture collection now consists of over 125,000 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 ARS. 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.
Resistance genes may be carried on specific mobile plasmids which may be associated with specific animal sources. One particular type of Salmonella, serotype Typhimurium, is found in diverse agricultural niches and can be recovered from many different animal sources as well as humans. Typhimurium bacteria can carry the genes responsible for antimicrobial resistance to the cephalosporin antimicrobials on a mobile piece of DNA called plasmids. In the U.S., the antimicrobial susceptibility of Salmonella is monitored by the National Antimicrobial Resistance Monitoring System (NARMS). In this study, we determined the antimicrobial resistance of Salmonella Typhimurium to cephalosporins. In 2008, 70 isolates (70/581; 12.0 %) (34 isolates from retail meats, 23 from food animals, and 13 from clinically ill humans) were resistant to cephalosporins. All of the isolates had the bla(CMY) gene which was found on a plasmid in 59 (84.3%) of these isolates. We also discovered that these plasmids were primarily of two different types, one identified as IncI1 and the other as IncA/C. Isolates originating from chickens or chicken products were primarily identified as having IncI1 plasmids (37/40; 92.5%) while all isolates from cattle (6/23;26.1%) exclusively had IncA/C plasmids. Antimicrobial susceptibility patterns within the isolates recovered from IncI1 or IncA/C plasmids were also very similar. This suggests that during a food borne outbreak investigation when the source of an infection is unknown, if the laboratory knows that it is Salmonella Typhimurium and characterizes the type of cephalosporin resistant gene, the type of plasmid and determines the antimicrobial resistance profile, they may be able to identify the food source of the outbreak. This is particularly important for physicians, veterinarians, public health laboratories, and epidemiologists as they respond to foodborne outbreaks.
Prevalence and antimicrobial resistance of Salmonella, E. coli, and Campylobacter in pigs from swine producing states in the United States. The development of antimicrobial resistance remains a significant concern when treatment is indicated. We determined the prevalence and antimicrobial susceptibility of Salmonella, Campylobacter and generic E. coli from swine feces collected over one year from the top three swine producing states (Iowa, North Carolina, and Minnesota), which represent 51% of the total pig crop in the U.S, plus Ohio. Up to 30 fresh fecal samples were collected per barn from a total of 148 barns across all states (n=4,426 samples); collections were divided by season. All bacteria were isolated using standard culture methods. Antimicrobial susceptibility testing was determined using broth microdilution; molecular fingerprinting was determined by Pulsed Field Gel Electrophoresis. The prevalence of Salmonella (n=462/4426), Campylobacter (n=994/1184) and E. coli (n=833/845) at the sample level was 10.4%, 98.6% and 83.6%, respectively. Overall, the top three Salmonella serotypes were Typhimurium (42%), Derby (25%) and Adelaide (5%); C. coli was the predominant Campylobacter species. Salmonella serotypes varied by barn within state and strain differences within serotypes by antibiogram and pulsotype were observed. Salmonella were most often resistant to Tetracycline (76%), Sulfisoxazole (59%), and Streptomycin (55%); however, serotype variation occurred. E. coli was most often resistant to Tetracycline (89%) and the Sulfonamides (33%); C. coli were most often resistant to Tetracycline (87%), Erythromycin (43%) and Azithromycin (43%). Less than 5.8% of E. coli and 5.6% of Salmonella were resistant to Ceftriaxone. Seasonal variations were observed. Further characterization of persistent versus non-persistent bacterial strains, including those that readily acquire resistance genes, may offer areas for development of mitigation strategies.
Characterization of Salmonella enterica serovar Dublin from cattle and humans. Salmonella Dublin is the second most common serotype isolated from cattle (2007 to 2011; animal arm of the National Antimicrobial Resistance Monitoring System (NARMS)), is among the most invasive serotypes in humans, and an increase in multi-drug resistance (MDR) has been observed. We determined the antimicrobial resistance, the presence of integrons and replicon types of plasmids in Dublin isolated from cattle and humans submitted to NARMS (1999-2011). Antimicrobial resistance was determined by broth microdilution for 272 cattle (C) and 69 human (H) isolates. Isolates were screened for integrons (class 1, 2 and.
Antimicrobial resistance to clinically important antimicrobials. Antimicrobial treatment is typically not required to treat Salmonella infections. However, when indicated, cephalosporins are drugs of choice. Bacteria evade killing by cephalosporins through use of internal enzymes called beta-lactamases. Three main groups of beta-lactamases among Salmonella are the cephamycinases (CMY), the carbapenemases and the extended spectrum beta-lactamases (ESBLs). In North America the CMY beta-lactames are prevalent and antimicrobial susceptibility of Salmonella is monitored by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) and The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). In this study, we determined the susceptibility to cephalosporins by broth microdilution among 5,045 non-Typhi Salmonella enterica isolated from food animals, retail meats and humans. In the United States, 109 (4.6%) isolates collected from humans, 77 (15.6%) from retail meats, and 140 (10.6%) from food animals displayed decreased susceptibility to cephalosporins (DSC). Among the Canadian retail meat and food animal isolates, 52 (13.0%) and 42 (9.4%), respectively displayed DSC. At least one beta-lactamase gene was detected in 74/109 (67.9%) isolates collected from humans and bla(CMY) genes were most prevalent (69/109;63.3%). Similarly, bla(CMY) genes predominated among the beta-lactamase-producing isolates collected from retail meats and food animals. ESBLs were found among three isolates originating from children who had been adopted from outside of North America. The overlap of bla(CMY) genes from the same type of Salmonella in food animals and humans suggests that food animals are reservoirs for the bacteria. Continued surveillance is warranted to monitor the emergence of beta-lactamase resistant bacteria as well as to implement mitigation strategies.
4)and incompatibility (Inc) replicon types using standard PCR protocols. Percent resistance was observed most often to: Ampicillin (63% C; 41% H), Ceftriaxone (19%C; 20%H), Chloramphenicol (60% C; 43% H), Kanamycin (51% C; 39% H), Streptomycin (69% C, 43% H), Sulfa antimicrobials (65% C; 49% H), and Tetracycline (67%C; 46% H). No resistance to Amikacin or Ciprofloxacin was observed. MDR (>=5 antimicrobials) was observed in 43% of both cattle and humans isolates. The most common MDR pattern in cattle was Amoxicillin – Clavulanic Acid/Ampicillin/Cefoxitin/Ceftiofur/Ceftriaxone/Chloramphenicol/Kanamycin/Streptomycin/Sulfa/Tetracyline (n=27) and Ampicillin/Chloramphenicol/Kanamycin/Streptomycin/Sulfa/Tetracycline (n=11) in humans. Only class 1 integrons were identified in 30% (n=82) and 25% (n=17) of the cattle and human isolates, respectively. The replicon types identified from all isolates were: FIIS (80% C; 91% H), IncA/C (52% C; 29% H), H1 (17% C;16% H), FIA (17% C; 3% H), I1 (7% C; 1% H), FIB (6% C; 3% H), P (3% C; 1% H) and N (1% C; 0% H ). The presence of integrons and Inc FIIS, Inc A/C, Inc I1 and Inc N may be useful in addition to susceptibility testing when studying the transmission of resistance genes and developing mitigation strategies.
Estimation of prevalence and comparison of Salmonella culture results from individual, pooled, and composite fecal samples for determining herd infection status and identifying serovar(s). During the U.S. Department of Agriculture National Animal Health Monitoring System Dairy 2007 study, data and samples were collected from dairy operations in 17 major dairy states. As part of the study, composite fecal samples (six per operation) were collected from cow areas, such as holding pens, alleyways, and lagoons, where manure accumulates. Fecal samples also were collected from individual cows (35 per operation), and fecal sample pools were created by combining samples from 5 cows (7 per operation). A total of 1,541 composite fecal samples were collected from 260 operations in 17 states, and 406 (26.3%) of these samples were culture positive for Salmonella. Among the 116 operations for which all three sample types were obtained, 41.4% (48 operations) were Salmonella culture positive based on individual samples, 39.7% (46 operations) were positive based on pooled samples, and 49.1% (57 operations) were positive based on composite fecal samples. Relative to individual samples, the sensitivity of composite fecal samples for determining herd infection status was 85.4% and the sensitivity of pooled fecal samples was 91.7%. On 33.6% of operations (39 of 116), Salmonella was cultured from all three fecal sample types (individual, pooled, and composite), and 20 (51.3%) of these operations had exactly the same serovar in all three sample types. Use of composite fecal samples is less costly and time-consuming than use of individual or pooled samples and provides similar results for detecting the presence and identifying serovars of Salmonella in dairy herds. Therefore, composite sampling may be an appropriate alternative to culture of individual samples when assessing Salmonella status in dairy herds.
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Cox Jr, N.A., Cason, J.A., Buhr, R.J., Richardson, K.E., Richardson, L.J., Rigsby, L.L., Cray, P.J. 2013. Variations in preenrichment pH of poultry feed and feed ingredients after incubation periods up to 48 hours. Journal of Applied Poultry Research. 22(2):190-195.
Folster, J.P., Pecic, G., Singh, A., Duval, B., Rickert, R., Ayers, S., Abbott, J., Mcglinchey, B., Bauer-Turpin, J., Haro, J.H., Hise, K., Zhao, S., Cray, P.J., Whichard, J., Mcdermott, P.F. 2012. Characterization of extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg isolated from food animals, retail meat, and humans in the United States 2009. Foodborne Pathogens and Disease. 9(7):638-645.
Lombard, J.E., Beam, A.L., Nifong, E.M., Fossler, C.P., Kopral, C.A., Dargatz, D.A., Wagner, B.A., Erdman, M.M., Cray, P.J. 2012. Comparison of individual, pooled, and composite fecal sampling methods for detection of Salmonella on U.S. dairy operations. Journal of Food Protection. 75(9):1562-1571.
Molla, B., Byrne, M., Abley, M.J., Matthews, J., Jackson, C.R., Cray, P.J., Sreevatsan, S., Wang, P., Wondwossen, G. 2012. Epidemiology and genotypic characteristics of Methicillin-Resistant Staphylococcus aureus strains of porcine origin. Journal of Clinical Microbiology. 50(11):3687-3693.
Sohn, M., Himmelsbach, D.S., Barton , F.E., Cray, P.J. 2009. Flourescence spectroscopy for rapid detection and classification of bacterial pathogens. Applied Spectroscopy. 63(11):1251-1255.