Cummings Lab Research Description, 2009
Dangerous DNA: Antibiotic Resistance Genes in the Natural Environment
Urban stormwater introduces numerous pollutants into California's coastal environment from
anthropogenic sources such as landscaping, city streets, accidental spills, and illegal waste
disposal. Coastal waters are often considered unsafe for human contact in the days following a
storm due to bacterial and viral pathogens originating from fecal sources. The public health
hazards are compounded in areas where human sewage disposal and treatment are inadequate.
Current management of stormwater in coastal waters entails monitoring fecal indicator bacteria
and restricting access until counts are considered safe. The assumption is that the primary
biological threat posed by stormwater is from fecal pathogens. Less obvious, but of potentially
great long-term concern, are antibiotic-resistant bacteria and the antibiotic resistance genes they
carry. Our recent work, funded by California Sea Grant, suggests that antibiotic-resistant bacteria
and their resistance genes are introduced to coastal wetlands by storm events, and that they may
be accumulating in sediments. It is well known that bacteria readily share portions of their
genomes, such as resistance genes, even in the absence of a selective pressure, a process known
as horizontal gene transfer. Thus, if resistant bacteria and resistance genes accumulate and persist
in coastal wetlands, these habitats could conceivably act as reservoirs of dangerous biological
pollutants. Responsible management of coastal wetland ecosystems in urban environments
requires an understanding of the nature and extent of these pollutants.
The Tijuana River Estuary
Famosa Slough
We primarily work in three coastal wetlands and one stream. The Tijuana River Estuary (N32o
33’ W117o 07’) is a 1,000-ha National Estuarine Research Reserve, National Wildlife Refuge,
and Wetland of International Importance (Ramsar Site #1452) on the US-Mexico border in
Imperial Beach, CA (San Diego County). The 4,400-km2 watershed, two-thirds of which lies in
northern Baja California, Mexico, includes urban areas (Tijuana, Tecate, and Imperial Beach),
agricultural land, and open chaparral and sage scrub. Raw sewage from Tijuana is disposed
directly into the River and routed into the Southbay International Wastewater Treatment Plant
during dry weather. However, during winter rains, the sewage-polluted stormwater is released
directly to the Estuary. Famosa Slough (N32o 45’ W117o 13’) is a small wetland (15 ha) in
urban San Diego. While Famosa Slough receives urban runoff from the greater San Diego area,
it has been relatively protected from sewage contamination. The Ballona Wetlands (N33o 58’
W118o 27’) are located about 2 km NW of Los Angeles International Airport, consisting of the
remaining 0.8 km2 of what was once a much larger coastal wetland. This watershed is highly
urbanized, home to over 1.5 million people, covers approximately 337 km2, and drains a
significant portion of the Los Angeles basin. Finally, Pine Valley Creek (N32o 52’ W116o 31’)
is an intermittent stream in the undeveloped mountains east of San Diego within the Tijuana
River watershed. The primary activities in the Pine Valley Creek sub-watershed are recreational
and sewage contamination is not known to be a problem.
We are taking two approaches to investigating this problem. The first approach entails
quantifying the extent of sediment contamination with antibiotic resistance genes using real-time
quantitative PCR (qPCR). Our current focus is on plasmid-mediated quinolone resistance
(PMQR) determinants, which provide bacteria with protection from drugs such as ciprofloxacin
(Cipro®) and levofloxacin (Levaquin®). PMQR determinants include the various Qnr
pentapeptide-repeat protein families that protect bacterial topoisomerases from quinolone drugs,
the quinolone acetylase enzyme AAC(6’)-Ib-cr, and a recently discovered quinolone-specific
efflux pump termed QepA. We hope to be able to determine the extent to which clinically
relevant antibiotic resistance genes have contaminated these urban wetland habitats.
In the second approach, we are isolating and characterizing multidrug-resistance plasmids,
mobile genetic elements encoding resistance to diverse classes of antibiotics, from the Tijuana
River Estuary. In the spotlight right now is a 45-kb plasmid called pLNU.11 from Citrobacter
freundii strain AtetA isolated from Tijuana River Estuary sediments after a winter storm.
pLNU.11 confers resistance to quinolone drugs, β-lactam antibiotics (e.g., penicillin,
cephalexin), tetracyclines, and sulfa drugs (e.g., sulfamethoxazole-trimethoprim). We hope to
determine the relative importance of this plasmid in the wetland, whether it is present at the other
sites, and whether it is readily transmissible to other species of bacteria.
Pieter Baker, Karisa Archer, Jonathan Laroya, Kelly Pfeil
Honors Theses
Fall-Spring 2008-2009
David Arriola (B.S. Biology-Chemistry with honors, 2009)
Honors thesis title: Quantification of antibiotic resistance genes in urban wetland sediments.
David won the Samuel and Sussanah Wesley Best Honors Scholarship Award for this project.
Student Researchers
All work in the Cummings Lab is carried out by undergraduate researchers majoring in Biology
or Biology-Chemistry at PLNU. The following students have contributed to research in the
Cummings Lab in the past 5 years:
2005 Amy Hebling, TJ Kopshy, and Nathan Singh,
2006 Grace Faucett, Amy Hebling, TJ Kopshy, Jason Kroening, Nathan Singh, and Kelsey
Unruh
2007 David Arriola, Grace Faucett, Jason Kroening, Cody Ryan, Nathan Singh, and Kelsey
Unruh
2008 Karisa Archer, David Arriola, Jonathan Laroya, Daniel Palmer, Cody Ryan, and
Kelsey Unruh
2009 Karisa Archer, David Arriola, Pieter Baker, Jonathan Laroya, and Kelly Pfeil
Undergraduate Presentations
1.
Unruh, K., D. Arriola, *C. Ryan, J. Laroya, K. Archer, and David Cummings. 2009.
The search for antibiotic-resistant bacteria in coastal wetlands using growth-based
methods. West Coast Biological Sciences Undergraduate Research Conference, San
Diego, CA. (oral presentation)
2.
Archer, K. F., *D. A. Arriola, J. B. Laroya, C. R. Ryan, N. J. Singh, K. R. Unruh,
and D. E. Cummings. 2009. Investigation of antibiotic resistance genes in urban wetland
sediments. West Coast Biological Sciences Undergraduate Research Conference, San
Diego, CA. (oral presentation)

David Arriola Won Best Student Presentation, Microbiology
3.
*Arriola, D. A., K. F. Archer, J. B. Laroya, C. R. Ryan, N. J. Singh, K. R. Unruh,
and D. E. Cummings. 2008. Antibiotic resistance genes in the metagenome of urban
wetland sediments. 16th Annual International Meeting on Microbial Genomics, Lake
Arrowhead, CA. (poster)
4.
*Unruh, K. R., C. Ryan, N. Singh, D. Arriola, and D. Cummings. 2008. The search
for antibiotic-resistant bacteria in coastal wetlands using DNA-based and growth-based
methods. West Coast Biological Sciences Undergraduate Research Conference, San
Diego, CA. (poster)
5.
*Unruh, K. R., A. E. Hebling, S. Spring, and D. E. Cummings. 2007. Microbial
diversity of native and Fe(III)-enriched consortia from the sediment-water interface of an
estuarine coastal wetland. American Society for Microbiology, General Meeting,
Toronto, Canada. (poster)
6.
*Unruh, K. R., A. E. Hebling, S. Spring, and D. E. Cummings. 2007. Microbial
diversity of native and Fe(III)-enriched consortia from the sediment-water interface of an
estuarine coastal wetland. West Coast Biological Sciences Undergraduate Research
Conference, Los Angeles, CA. (poster)
7.
*Hebling, A., and D. E. Cummings. 2006. Exploratory research on Fe(III)-reducing
bacteria in the sediments of the Tijuana Estuary. West Coast Biological Sciences
Undergraduate Research Conference, San Diego, CA. (oral presentation)
8.
*Kopshy, T. J., and D. E. Cummings. 2006. Reduction of chromium (VI) by
Acidiphilium cryptum. West Coast Biological Sciences Undergraduate Research
Conference, San Diego, CA. (poster)
9.
*Singh, N., T. J. Kopshy, and D. E. Cummings. 2006. Evidence for enzymatic
reduction of Cr(VI) by Acidiphilium cryptum. West Coast Biological Sciences
Undergraduate Research Conference, San Diego, CA. (poster)
* Presenting Author