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Prevalence and Seasonality of Antibiotic Resistant Bacteria in the Coastal Environment Project

Summary

The CDC has recently released a report that calls antibiotic resistance (AR) “one of our most serious health threats” (CDC 2013 Report).  This is a result of current antibiotics becoming less effective and the fact that new antibiotics are not being created, which makes understanding the spread of resistance all the more critical (Yang 2013).  Even though it is widely accepted as a critical issue, relatively little research is being conducted regarding environmental reservoirs of AR, particularly in the marine environment.  Because of this, many seemingly basic questions are left unanswered.  Some of this unknown basic data includes the level of antibiotics present within the coastal environment and the level of antibiotic resistance bacteria existing naturally as well as present due to human impacts on the environment.  This dearth of knowledge is particularly surprising when you recognize the amount of antibiotics that are utilized within the United States by humans, livestock, and other agricultural applications. Eventually, these products make their way from their original uses in aquaculture, agriculture, hospitals, and human bodies into the ocean, where they transfer AR genes to marine bacterial reservoirs or vice versa (Yang 2013).

Previous research has illustrated that antibiotic resistant bacteria (ARB) present in estuaries in urban landscapes may be tied to wet weather sewage (Young 2013).  Yet, this study only examined heterotrophic bacteria and studied 2 antibiotics (Young 2013).  Research by the Gast lab has illustrated the high levels of antibiotic resistance that are present in marine animals nearby Cape Cod; however research has not yet examined levels within coastal waters, sand and invertebrates in this area (Rose 2009).  As people recreate in the coastal systems - on the water and on the beach -and as people consume food products that live in these ecosystems, the potential of interacting with an antibiotic resistant strain of bacteria becomes a serious issue.  The goal of this study is to provide a preliminary seasonal understanding of the distribution of antibiotic resistance at local beaches in relation to human activity.

This study will focus on three sites that have varying levels of direct human activity instead of agricultural and hospital wastes.  Little Island is a low impact site because it is located within a residential development and is relatively isolated from the presence of humans.  Waquoit Bay and Old Silver Beach are two high impact sites.  Waquoit Bay is a seasonally eutrophied water body, with a high density of residences that may contribute the eutrophication through land-based runoff and septic systems.  Old Silver Beach is an extremely popular swimming area, especially during the summer. Sampling would occur every three months over a period of 1 year to capture the influence of season on bacterial abundance, diversity, and antibiotic resistance.  At each location, water, sand, and oysters will be collected to culture heterotrophic marine bacteria, Enterococcus, and Vibrio. These bacteria have been chosen specifically because of their potential roles as reservoirs of antibiotic resistance.  Heterotrophic bacteria will be examined as a general survey of marine bacteria that may serve to harbor AR genes.  Enterococci have been chosen as they are utilized to monitor possible fecal contamination in the beach environment.  Finally, Vibrio species are potentially pathogenic to humans and therefore indicate an important public health concern. For each bacterial type, a maximum of 20 bacterial colonies will be chosen per environmental sample and a maximum of 10 colonies per oyster sample.  Recovery of colonies can vary and in general 50-75% of chosen colonies actually begin growing. Then, these colonies will be used to determine antibiotic resistance, as each sample will be tested for sensitivity to azithromycin, amoxicillin, ciprofloxacin, clindamycin, doxycycline, oxytetracycline and trimethoprim.

From this preliminary work, I will determine the prevalence of antibiotic resistance in three different groups of bacteria, and will uncover insights into the seasonal persistence of resistance.  I expect that the prevalence of ARB will be higher at Old Silver Beach and Waquoit Bay than at Little Island, and presume that ARB will decline at all sites during the winter.  The information will be used to test whether the level of human activity correlates to the prevalence and/or persistence of environmental antibiotic resistance within a coastal system.  Furthermore, this study has public health impacts to determine what potential risks are associated from beach recreation and shellfish consumption.

Funding

A Coastal Ocean Institute grant for this study would go directly towards purchasing supplies for this project -additional antibiotic disks and components for enterococci media.  To cover this, I am requesting $1,267.  Currently, I have an NSF Graduate Fellowship that supports my salary and part of my tuition, but there are only limited research supply funds to support this project.

Relevance to thesis

I am broadly interested in microbiology, oceanography, public health, and microbial biogeography.  Environmental antibiotic resistance, to me, connects all of these interests together and is what I plan to pursue for my thesis project.  As a first-year student, this is my first experiment upon which I will build the rest of my thesis work. 

In later experiments, I plan to broaden the scope to look at agricultural, hospital, and wastewater treatment wastes to determine what anthropogenic sources are the largest contributors to antibiotic resistance and whether patterns of resistance to particular antibiotics can implicate the source of contamination. If possible, it would be beneficial to study this in multiple parts of the world – or at least on different US coasts – to see how regional differences in antibiotic use impact the resistance profiles found in the environment.

Another aspect to my project is the determination of rates of antibiotic resistance acquisition and transfer. I plan on setting up marine mesocosms that would allow examination of AR establishment relative to anthropogenic inputs.  Prior research by Engemann examined the fate of tetracycline resistance in mesocosms in artificial ponds that were supplemented with cattle waste additions (2008).  To my knowledge, the use of mesocosms to tease apart AR levels in the marine environment would be a novel approach.  Receiving this grant would give me supply support to begin this antibiotic resistance research that will be the crux of my thesis and will serve as the preliminary data for a proposal to NIH. 

References

Center for Disease Control and Prevention. Antibiotic resistance threats in the United States, 2013. 1–114 (2013). Yang, J. et al. Marine sediment bacteria harbor antibiotic resistance genes highly similar to those found in human

pathogens. Microb. Ecol. 65, 975–81 (2013). Young, Suzanne; Johl, A. & O. Mullan, G. D. Antibiotic-resistant bacteria in the Hudson River Estuary linked to

wet weather sewage contamination. J. Water Health 11, 297–310 (2013). Rose, J. M. et al. Occurrence and patterns of antibiotic resistance in vertebrates off the Northeastern United States

coast. FEMS Microbiol. Ecol. 67, 421–31 (2009). Engemann, C. a, Keen, P. L., Knapp, C. W., Hall, K. J. & Graham, D. W. Fate of tetracycline resistance genes in

aquatic systems: migration from the water column to peripheral biofilms. Environ. Sci. Technol. 42, 5131–6 (2008).

Last updated: December 3, 2013