David M. Wagner1, Janelle Runberg1, Amy J. Vogler1, Judy Lee1, Lance B. Price2, David M. Engelthaler2,

Jacques Ravel3, & Paul Keim1

 1Northern Arizona University, Flagstaff, AZ;

2Translational Genomics Research Institute (TGen), Flagstaff, AZ; 3University of Maryland School of

Medicine, Baltimore, MD

No evidence of plasmid-mediated antibiotic resistance

in North American Yersinia pestis

Plague Transmission Cycle

Pathways

usual

occasionalrare or

theoreticalCommensal Rat Cycle

InfectiveFlea

InfectiveFlea

DomesticRodent

DomesticRodent

Dire

ct

co

nta

ct

WildRodent

WildRodent

InfectiveFlea

InfectiveFlea

Direct contact

Wild Rodent Cycle

Bubonicor Septicemic

plague

Secondaryplague

pneumonia

Dir

ec

t c

on

tac

t

co

nta

min

ate

d s

oil

Primarypneumonic

plaguecases

Slide courtesy of Ken Gage

Three Plague Pandemics – 200 Million Deaths

Perry & Fetherston 1997Achtman et al. 1999

Control of Plague – Hygiene & Antibiotics

Kill or eliminate habitat for rat hosts – very important in urban areas

Control flea vectors using insecticidesRapid diagnosis, followed byStandard treatment with antibiotics

Streptomycin Tetracyclines Sulfonamides

Plague Today – Global Distribution

Stenseth et al. 2008. PLoS Medicine

Plague Today – Increases in Africa

Stenseth et al. 2008. PLoS Medicine

Plague Today – Increases in Africa

Stenseth et al. 2008. PLoS Medicine

Plague Vaccines – Little Success to Date

Killed vaccine no longer available in the USLive attenuated vaccine not licensed for humansInjected subunit vaccines show promise for futureAs 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

Strain 17/95 16/95

Streptomycin Resistance

YES YES

Tetracycline Resistance

YES NO

Sulfonamide Resistance

YES NO

Other Resistance YES NO

Country of Origin Madagascar Madagascar

Year of Isolation 1995 1995

Resistance Plasmid pIP1202 pIP1203

Galimand et al. 1997. 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. 2001 ; Prentice et al. 2001

Plasmid Acquisition Likely Occurs in Fleas

In co-infected fleas, E. coli donated pIP1202 to Y. pestis at frequency of 10-3 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. 2007. 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, porkStates: CA, CO, CT, GA, IA, MD, MN, ND, NM, NY,

TN, and ORHosts: S. enterica Typhimurium, Newport, Kentucky,

Heidelberg, Dublin, Bredeney, Klebsiella spp., E. coliMost resistant to tetracycline and many resistant to

streptomycin and others, in addition to sulfonamidesMany strains readily transferred plasmids to Y.

ruckeri

Welch et al. 2007. PLoS One

No Evidence in North American Y. pestis

State N Years

Arizona 151 1975, 1977-1984, 1986-1989, 1992-1996, 1998, 2000-2002

California 129 1943, 1962, 1970, 1977, 1979-1980, 1983-1999

Colorado 97 1963, 1968, 1989, 1992, 1995-1997, 1999-2002

Idaho 2 1987, 1997

Kansas 17 1997, 1999

Montana 11 1987, 1992-1993

North Dakota 2 1986, 1993

New Mexico 124 1950, 1976-1977, 1979-1988, 1991-1992, 1994-1995, 1997-2002

Nevada 36 1980-1985, 1987, 1992-1995

Oregon 18 1959, 1970-1971, 1977, 1979, 1981-1984, 1987

Texas 5 unknown

Utah 55 1965, 1981-1984, 1991-1995, 1999-2001

Washington 2 1984

Wyoming 64 1978, 1980, 1982-1983, 1985-1987, 1989-1990, 1997, 2000-2001

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 LevyCoconino County Health Department: Marlene

GaitherNAU Y. pestis Group: Amy Vogler, Becky Colman,

Joe Busch, Judy Lee, Adina Doyle, Roxanne Nera