David M. Wagner 1, Janelle Runberg 1, Amy J. Vogler 1, Judy Lee 1, Lance B. Price 2, David M. Engelthaler 2, Jacques Ravel 3, & Paul Keim 1 1 Northern Arizona University, Flagstaff, AZ; 2 Translational Genomics Research Institute (TGen), Flagstaff, AZ; 3 University of Maryland School of Medicine, Baltimore, MD No evidence of plasmid-mediated antibiotic resistance in North American Yersinia pestis

Plague Transmission Cycle Pathways usual occasional rare or theoretical Commensal Rat Cycle Infective Flea Infective Flea Domestic Rodent Domestic Rodent Direct contact Wild Rodent Wild Rodent Infective Flea Infective Flea Direct contact Wild Rodent Cycle Bubonic or Septicemic plague Secondary plague pneumonia Direct contact contaminated soil Primary pneumonic plague cases Slide courtesy of Ken Gage

Three Plague Pandemics – 200 Million Deaths Perry & Fetherston 1997 Achtman et al. 1999

Control of Plague – Hygiene & Antibiotics Kill or eliminate habitat for rat hosts – very important in urban areas Control flea vectors using insecticides Rapid diagnosis, followed by Standard treatment with antibiotics  Streptomycin  Tetracyclines  Sulfonamides

Plague Today – Global Distribution Stenseth et al PLoS Medicine

Plague Today – Increases in Africa Stenseth et al PLoS Medicine

Plague Today – Increases in Africa Stenseth et al PLoS Medicine

Plague Vaccines – Little Success to Date Killed vaccine no longer available in the US Live attenuated vaccine not licensed for humans Injected subunit vaccines show promise for future As a result, efforts to save human lives are still focused on rapid diagnosis followed by treatment with antibiotics Resistance to antibiotics could represent a serious threat to human health given high pathogenicity and ability to rapidly spread under optimal conditions

Antimicrobial Resistance in Y. pestis Strain17/9516/95 Streptomycin ResistanceYES Tetracycline ResistanceYESNO Sulfonamide ResistanceYESNO Other ResistanceYESNO Country of OriginMadagascar Year of Isolation1995 Resistance PlasmidpIP1202pIP1203 Galimand et al New England Journal of Medicine

Typical and Atypical Plasmids in Y. pestis Three main plasmids, all associated w/ virulence:  pCD1 (found in all pathogenic Yersinia)  pPCP1  pMT1 pMT1 very similar to plasmid in Salmonella enterica Serovar Typhi Several different studies have documented atypical plasmids present in Y. pestis strains Indicates that this species readily acquires plasmids Filippov et al. 1990; Parkhill et al ; Prentice et al. 2001

Plasmid Acquisition Likely Occurs in Fleas In co-infected fleas, E. coli donated pIP1202 to Y. pestis at frequency of after three days After four weeks, 95% of co-infected fleas contained MDR Y. pestis (Hinnebusch et al. 2002) Y. pestis infected fleas can harbor diverse bacterial communities (Wagner et al. unpubl.) Y. pestis infected fleas commonly co-infected with Salmonella spp. (Eskey et al. 1951) What do we know about MDR plasmids in Y. pestis?

Plasmid pIP1202 from Y. pestis Similar to MDR plasmids from Y. ruckeri and S. enterica Newport All share the same plasmid backbone (IncA/C) Backbone contains gene conferring resistance to sulfonamides (sul2) Other resistance genes vary Welch et al PLoS One

Similar MDR Plasmids in US Meat Products Plasmids with similar IncA/C backbones and varying MDR profiles found in bacteria recovered from meat Sources: turkey, chicken, beef, pork States: CA, CO, CT, GA, IA, MD, MN, ND, NM, NY, TN, and OR Hosts: S. enterica Typhimurium, Newport, Kentucky, Heidelberg, Dublin, Bredeney, Klebsiella spp., E. coli Most resistant to tetracycline and many resistant to streptomycin and others, in addition to sulfonamides Many strains readily transferred plasmids to Y. ruckeri Welch et al PLoS One

No Evidence in North American Y. pestis StateNYears Arizona , , , , 1998, California , 1962, 1970, 1977, , Colorado971963, 1968, 1989, 1992, , Idaho21987, 1997 Kansas171997, 1999 Montana111987, North Dakota21986, 1993 New Mexico , , , , , Nevada , 1987, Oregon181959, , 1977, 1979, , 1987 Texas5unknown Utah551965, , , Washington21984 Wyoming641978, 1980, , , , 1997,

Discussion/Conclusions No IncA/C plasmid-mediated MDR in North American Y. pestis – why? Our isolates mostly from human plague investigations MDR resistant plasmids in meats probably arose in Concentrated Animal Feeding Operations (CAFOs) Plague limited to 17 westernmost states, whereas most CAFOs in the eastern states Plausible that MDR Y. pestis could arise in fleas co- infected with MDR enteric pathogens and Y. pestis However, no obvious selection pressure to maintain these MDR plasmids in Y. pestis

Acknowledgements Funding: NIH-NIAID, NIH Pacific-Southwest Regional Center of Excellence, Arizona Game & Fish, NAU- Cowden Endowment CDC-Ft. Collins: Ken Gage, Becky Eisen, Jeannine Petersen, Marty Schriefer, Michael Kosoy Arizona Department of Health Services: Craig Levy Coconino County Health Department: Marlene Gaither NAU Y. pestis Group: Amy Vogler, Becky Colman, Joe Busch, Judy Lee, Adina Doyle, Roxanne Nera