Antimicrobial resistance trends in northern California dairy cattle Salmonella isolates, 2002-2016 Background Objective Results Discussion Acknowledgements References • Nontyphoidal Salmonella infections contribute to 1.2 million annual illnesses in the United States 1 • Salmonella is a significant foodborne pathogen; human outbreaks have been associated with dairy products, ground beef, and contact with dairy cattle 2,3,4 • Salmonella antimicrobial resistance (AMR) is a serious concern that can increase recovery time, medical costs, and mortality rates in humans and animals 1 • Surveillance data of AMR in Salmonella isolated from cattle can aid in improving treatment plans, managing current AMR, and preventing future AMR development Identify trends in AMR phenotype of Salmonella isolates obtained from dairy cattle fecal samples at the University of California, Davis Veterinary Medical Teaching Hospital microbiology laboratory from 2002 to 2016 Kelly E. Davidson*, Richard V. Pereira 1 , Barbara A. Byrne 2 University of California, Davis • School of Veterinary Medicine • Population Health and Reproduction 1 • Pathology, Microbiology & Immunology 2 • Multidrug resistance (MDR) was observed in 50.8% of Salmonella isolates • Three, similar MDR patterns were observed in 29.3% of Salmonella isolates. The most common was a pattern of nine antimicrobials (Table 1). The second and third most common patterns included the same nine drugs in addition to either trimethoprim/sulfisoxazole or gentamicin. • Significantly higher odds for resistance to aminoglycosides, beta-lactam/beta-lactamase inhibitor combinations, folate pathway inhibitors, penicillins, and tetracyclines were observed for the 2002-2009 period when compared to the 2010-2016 period • Cephalosporins, quinolones, and macrolides (all antimicrobial classes containing medically important drugs) did not show a significant change in AMR between the 2002-2009 and 2010-2016 periods. Boehringer-Ingelheim Veterinary Scholars Program U.S. Department of Agriculture Project number CA-V-PHR-4069-H “Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture” University of California, Davis Veterinary Medical Teaching Hospital Microbiology Laboratory Conclusion 1 Antibiotic Resistance Threats in the United States, 2013. 71-72 (Centers for Disease Control and Prevention, 2013). 2 Gould, L. H., Mungai, E. & Behravesh, C. B. Outbreaks attributed to cheese: differences between outbreaks caused by unpasteurized and pasteurized dairy products, United States, 1998-2011. Foodborne Pathog Dis 11, 545-551, doi:10.1089/fpd.2013.1650 (2014). 3 Multistate Outbreak of Salmonella Typhimurium Infections Linked to Ground Beef (Final Update), <https://www.cdc.gov/salmonella/2011/ground-beef-2-1-2012.html> (2011). 4 Multistate Outbreak of Multidrug-Resistant Salmonella Heidelberg Infections Linked to Contact with Dairy Bull Calves (Final Update), <https://www.cdc.gov/salmonella/heidelberg-11-16/index.html> (2017). 5 OPS Diagnostics. Available at http://opsdiagnostics.com/images/products/1253.jpg. Accessed July 22, 2017. 6 Avena Medica. Available at http://www.avena-medica.com/ProductVault/product_1351076952__mg_3625_S4.jpg. Accessed July 22, 2017. 7 Microbe Online. Available at http://microbeonline.com/types-of-bacteriological-culture-medium/. Accessed July 22, 2017. 8 Karter Scientific. Available at https://www.kartersci.com/16x100mm_Test_Tubes_Borosilicate_Glass_Round_Btm_p/402f2.htm. Accessed July 22, 2017. 9 EdgeBio. Available at https://www.edgebio.com/sites/default/files/96-Well%20Treated%20Microplates%20V-bottom.jpg. Accessed July 22, 2017. 10 Bruker. Available at https://www.bruker.com/fileadmin/_processed_/csm_microflex-lt-product-banner_1d453de04d.jpg. Accessed July 22, 2017. Despite reduced odds of AMR to many drug classes in the 2010-2016 period, lack of a significant reduction in AMR for important drug classes such as cephalosporins, quinolones, and macrolides highlight the relevance of continual AMR surveillance in cattle with Salmonella infections to target future interventions. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Nonsusceptibility Prevalence Gentamicin Streptomycin 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Nonsusceptibility Prevalence Amoxicillin/clavulanic acid Ampicillin Cefoxitin Ceftiofur Ceftriaxone Nalidixic Acid 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Nonsusceptibility Prevalence Sulfisoxazole Trimethoprim/sulfamethoxazole 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Nonsusceptibility Prevalence Chloramphenicol Tetracycline Figure 1. Nonsusceptibility prevalance to aminoglycoside antimicrobials exhibits a decreasing linear trend from 2002-2016. There are significantly higher odds for nonsusceptibility to aminoglycosides for the 2002-2009 period when compared to the 2010- 2016 period (OR: 2.03, 95% CI: 1.1 – 3.7). Figure 2. Nonsusceptibility prevalence to nalidixic acid is minimal, and nonsusceptibility prevalence to other beta-lactam antimicrobials and a beta-lactam/beta-lactamase inhibitor combination exhibit decreasing linear trends from 2002-2016. There are significantly higher odds for nonsusceptibility to amoxicillin/clavulanic acid (OR: 1.79, 95% CI: 1.0 – 3.4) and ampicillin (OR: 1.87, 95% CI: 1.0 – 3.5) for the 2002-2009 period when compared to the 2010-2016 period. There is no significant difference in odds for nonsusceptibility to cephems for the 2002-2009 period when compared to the 2010-2016 period (OR: 1.65, 95% CI: 0.9 – 3.1). Nonsusceptibility to nalidixic acid occurs only in the 2010-2016 period. Figure 3. Nonsusceptibility prevalence to folate pathway inhibitors exhibits a decreasing linear trend from 2002-2016. There are significantly higher odds for nonsusceptibility to folate pathway inhibitors for the 2002-2009 period when compared to the 2010-2016 period (OR: 2.45, 95% CI: 1.3 – 4.5). Figure 4. Nonsusceptibility prevalence to chloramphenicol and tetracycline exhibit decreasing linear trends from 2002-2016. There are significantly higher odds for nonsusceptibility to tetracycline for the 2002-2009 period when compared to the 2010-2016 period (OR: 1.87, 95% CI: 1.0 – 3.4). There is no significant difference in odds for nonsusceptibility to chloramphenicol for the 2002-2009 period when compared to the 2010- 2016 period (OR: 1.54, 95% CI: 0.8 – 2.8). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Figure 5. Salmonella serotypes Dublin, Newport, and Typhimurium display nonsuceptibility to ceftriaxone in multiple years of the study period. Serotypes Newport, Dublin, and Typhimurium are also the most common serotypes observed throughout the study period, making up 21.5%, 19.0%, and 9.1% of serotypes, respectively. Table 1. Susceptibility patterns of 242 Salmonella isolates to the NARMS † gram-negative antimicrobial panel are varied, with nonsusceptibility to ceftriaxone observed in 40.1% of isolates. Au = amoxicillin/clavulanic acid A = ampicillin F = cefoxitin X = ceftiofur Cx = ceftriaxone C = chloramphenicol G = gentamicin N = nalidixic acid S = streptomicin Su = sulfisoxazole T = tetracycline T/S = trimethoprim/sulfimethoxazole All isolates were susceptible to azithromycin and ciprofloxacin. Methods Plates read using Sensititre Vizion System ® 24-hour, CO 2 incubation 18-hour, non- CO 2 incubation Inoculate 2-3 colonies 4-6-hour, non- CO 2 incubation Inoculate MH broth Inoculate Inoculate 18-hour, non- CO 2 incubation MALDI-TOF individual colony ≥ 24 hours old Isolation streaking frozen stabilates of Salmonella isolates on blood agar Plate on blood agar as QC * Serotyping of Salmonella isolates conducted using standard protocols by National Veterinary Services Laboratories in Ames, Iowa. Antimicrobial susceptibility testing followed Clinical Laboratory Standards Institute (CLSI) guidelines. References for photos: 5, 6, 7, 8, 9, 10; * QC = quality control; † NARMS = National Antimicrobial Resistance Monitoring System Susceptibility Pattern Count Prevalence Pansusceptible 78 0.322 AuAFXCxCSSuT 38 0.157 Su 34 0.141 AuAFXCxCSSuTT/S 17 0.070 AuAFXCxCGSSuT 16 0.066 AuAFXCxSSuTT/S 10 0.041 ASSuT 8 0.033 CSSuT 8 0.033 AuAFXCxCGSSuTT/S 7 0.029 ACSSuT 6 0.025 AuAFXCxCGSuT 6 0.025 S 6 0.025 ACGSSuTT/S 2 0.008 AuAFXCxCNSSuT 1 0.004 AuAFXCxCNSSuTT/S 1 0.004 AuAFXCxSSu 1 0.004 CGSSuT 1 0.004 FCGSSuTT/S 1 0.004 SSu 1 0.004 Total 242 1 Sterile saline BHI broth BHI broth NARMS † , gram-negative panel