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Characterization of Natural and Affected Environments
Antibiotic resistant bacteria in municipal wastes: Is there reason for concern? Ian Pepper, John P. Brooks, and Charles P. Gerba Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04360 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018
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Antibiotic resistant bacteria in municipal wastes: Is there reason for concern?
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Ian L. Peppera*, John P. Brooksb, and Charles P. Gerbaa
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W. Calle Agua Nueva, Tucson, AZ 85745;
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b
Water and Energy Sustainable Technology Center (WEST), The University of Arizona, 2959
Genetics and Sustainable Agriculture Research Unit, USDA ARS, Mississippi State, MS, 39762
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Corresponding author: *Ian L. Pepper
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Water and Energy Sustainable Technology Center (WEST, The University of Arizona, 2959 W.
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Calle Agua Nueva, Tucson, AZ 85745. E-Mail:
[email protected]. Ph: (520) 626-
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3328; Fax: 520-621-1032.
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Abstract
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Recently, there has been increased concern about the presence of antibiotic resistant bacteria
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(ARB) and antibiotic resistant genes (ARG), in treated domestic wastewaters, animal manures
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and municipal biosolids. The concern is whether these additional sources of ARB contribute to
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antibiotic resistance levels in the environment, i.e. “environmental antibiotic resistance.” ARB
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and ARG occur naturally in soil and water, and it remains unclear whether the introduction of
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ARB in liquid and solid municipal and animal wastes via land application have any significant
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impact on the background levels of antibiotic resistance in the environment, and whether they
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affect human exposure to ARB. In this current review, we examine and re-evaluate the incidence
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of ARB and ARG resulting from land application activities, and offer a new perspective on the
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threat of antibiotic resistance to public health via exposure from non-clinical environmental
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sources. Based on inputs of ARBs and ARGs from land application, their fate in soil due to soil
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microbial ecology principles, and background indigenous levels of ARBs and ARGs already
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present in soil, we conclude that while antibiotic resistance levels in soil are increased temporally
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by land application of wastes, their persistence is not guaranteed and is in fact variable, and often
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contradictory based on application site. Furthermore, the application of wastes may not produce
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the most direct impact of ARGs and ARB on public health. Further investigation is still
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warranted in agriculture and public health, including continued scrutiny of antibiotic use in both
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sectors.
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I.
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Microorganisms naturally produce and secrete antibiotic compounds, which can inhibit the
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growth of competing microbes in the environment.1 Due in part to their germicidal effects,
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antibiotics are useful as therapeutic agents for the control of infectious disease in humans and
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animals. The first and perhaps most effective antibiotic discovered was penicillin,
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serendipitously isolated from the soil fungus Penicillium by Sir Alexander Fleming in 1929. A
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second major antibiotic, streptomycin, was later isolated from the soil actinomycete
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Streptomyces griseus, by the soil microbiologist Selman Waksman in 1943. Waksman received
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the Nobel Prize for this discovery in 1952 - the only soil scientist to ever achieve such an honor.
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These powerful antibiotics revolutionized our ability to treat infectious diseases, but not without
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a concomitant cost.
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RECENT CONCERNS ON ARB AND ARG
Bacteria are simple prokaryotic microbes that can metabolize and replicate very quickly,
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resulting in remarkable genetic plasticity and adaptability. The existence of only one bacterial
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cell with a genetic or mutational change that confers resistance to an antibiotic agent encountered
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in the environment is sufficient for the subsequent proliferation of antibiotic resistant bacteria
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(ARB) that contain antibiotic resistant genes (ARG). ARGs are now considered to be a class of
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emerging contaminants.2 Thus, the more that antibiotics are used for the treatment or prevention
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of disease, the greater the likelihood that resistant strains will occur due to the selective
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advantages conferred by ARG. More specifically, the potential for the transfer of antibiotic
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resistance to human pathogenic microbes that subsequently can no longer be controlled by the
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prescribed antibiotics is of paramount concern. The conundrum then, is the more an antibiotic is
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utilized to prevent infectious disease, the less effective it will become over time. In addition,
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public health risks increase significantly when bacteria accumulate resistance to multiple
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antibiotics, making them particularly difficult to control as in the case of methicillin-resistant
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Staphylococcus aureus (MRSA). CDC estimates of antibiotic resistant infections are a minimum
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of 2 million3 people in the United States annually resulting in 23,000 deaths.
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ARB and ARG are commonly detected in domestic wastewater, agricultural waste releases
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from concentrated animal feedlot operations (CAFOs), biosolids and animal manures, hospital
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waste discharged into sewers, and soil and water.4 The role of anthropogenic activity on the
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incidence of ARB and ARG led to the introduction of the term “environmental antibiotic
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resistance,”5 as well as several review articles including “The Scourge of Antibiotic Resistance;
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The Important Role of the Environment,”6 and “Urban Wastewater Treatment Plants as Hotspots
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for Antibiotic Resistant Bacteria and Genes Spread into the Environment: A Review”.7 In this
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current review, we examine and re-evaluate the incidence of ARB and ARG resulting from land
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application activities, and offer a new perspective on the threat of antibiotic resistance to public
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health via exposure from non-clinical environmental sources.
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Specifically we discuss the relative incidence of ARBs in different anthropogenic-impacted
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environments relative to the naturally occurring level of incidence found in soils. Based on these
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data, we evaluate the impact of land application of biosolids, municipal effluents and animal
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manures on these natural levels found in soils. We also identify data gaps on pathogenic ARB
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characteristics that need to be identified before a formal risk assessment for antibiotic resistant
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pathogens can be performed. Overall, this review is meant to demonstrate the relative impact of
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anthropogenic activities versus “naturally” occurring soil-borne ARBs and ARGs.
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II.
INCIDENCE OF ARBs IN THE ENVIRONMENT, FOODSTUFFS, AND CLINICAL SETTINGS
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The discovery and implementation of antibiotics to aid in the fight against infectious diseases
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was a landmark event of great significance to public health. Yet, it also marked the rise of
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antibiotic-resistant microorganisms in health care settings, raising serious concerns as to the
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appropriate use of antibiotics in the treatment of infectious disease. Humans are exposed to
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millions of microbes with antibiotic resistant traits every day via natural and necessary activities
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such as the ingestion of water and food.8,9 However, the widespread use of antibiotics in the
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medical and agricultural industries has created an artificial selective pressure for the survival of
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ARB in some environments. This is especially true in health care environments, where
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antibiotics are routinely used to control the proliferation of human pathogens.10 In the clinical
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environment, the survival and transmission of ARB and ARG is a major concern due to the
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potential impacts on therapeutic outcomes. The importance of ARB and ARG outside of the
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clinical environment could be determined by employing a risk assessment approach that
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considers exposure, the nature of the pathogen, dose response, persistence in the environment,
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and the potential for genetic exchange.11 However for many of these parameters, data are
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lacking such that formal risk assessments do not exist. It is also important to consider the risk
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from exposure due to the environment relative to exposure to ARB and ARG originating from
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clinical environments.
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A.
ARBs and ARGs in Soil, Water and Residual Waste Materials
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In order to evaluate whether the occurrence of antibiotic resistance in the environment is actually
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a public health risk, multiple data sets are needed. These include the background or incidence
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concentrations of naturally-occurring ARB in the external environment where anthropogenic
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inputs of antibiotics are minimal or non-existent (e.g. undisturbed pristine soils), and also the
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levels of ARB introduced into soils due to anthropogenic activities including land application of
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biosolids, municipal effluents or animal manures. Here, antibiotic presence and ARB
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concentrations in different environments are considered.
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a) Soils
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The vast majority of antibiotics are natural products synthesized by soil microorganisms.12
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These include antibiotics effective against Gram-positive bacteria (e.g. penicillin), Gram-
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negative bacteria (e.g. polymixin), and the broad-spectrum antibiotics that are effective against
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both Gram-positive and -negative bacteria (e.g. chloramphenicol). These antibiotics are produced
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by diverse populations of soil microorganisms including bacteria, fungi and actinomycetes.13
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These natural products can be utilized by indigenous soil microbes as a form of self-defense
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against neighboring soil microorganisms. Interestingly, the gene clusters that result in antibiotic
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production are always present in the soil environment, but are only expressed under very specific
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conditions, such as interactions with other microbes.14
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Two perspectives on antibiotic resistance in soils are now well documented in the
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literature. The first is that antibiotic resistance is an ancient microbial attribute that existed on
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earth thousands or even billions of years before Fleming discovered the first antibiotic in 1929.
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Adu-Oppong et al.15 state that antimicrobials have likely been naturally produced by
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environmental microbes as a means of communication and defense for billions of years. For
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example, daptomycin, a clinically useful lipopeptide antibiotic produced by Streptomyces
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roseosporus may have evolved over 1 billion years ago16 Relatively more recently, D’Costa et
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al.17 concluded that analysis of Beringian permafrost sediments document that antibiotic
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resistance genes existed 30,000 years ago, showing conclusively “that antibiotic resistance is a
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natural phenomenon that predates the modern selective pressure of clinical antibiotic use.”
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The second documented perspective is that antibiotic resistance can be found in pristine areas
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unimpacted by anthropogenic activities. Recently, Durso et al.18 surveyed ungrazed, native
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prairie soil throughout Nebraska and showed ARG copy numbers ranging from ~103 to 105
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copies/g for tetracycline and sulfonamide resistance. Rahman et al.19 documented the occurrence
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of natural tetracycline resistance within bacteria located in marine sediments offshore of Japan.
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Diaz et al.20 identified antibiotic resistance genes in three different habitats across a permafrost
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thaw gradient within a pristine Arctic wetland. Taylor et al.21 found amphotericin B resistance in
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fungal Aspergilus spp. isolated from pristine caves in Brazil. Tetracylcine-resistant bacteria and
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resistance gene tet(M) were identified in fecal material from penguins in Antarctica.22 The
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concept of soil as a natural source of ARBs and ARGs was endorsed by Cytryn23 who eloquently
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stated that: “Although some evidence correlates between anthropogenic factors and elevated
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levels of antibiotic resistance in soil, it is becoming increasingly clear that unimpacted and
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pristine soils contain highly diverse and abundant levels of antibiotic resistant bacteria which
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harbor a wide array of clinically associated and novel antibiotic resistant genes.”
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The most prevalent biological entity on Earth (other than viruses) are bacteria, with recent
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estimates of total numbers on the order of 5 x 1030, the vast majority of which are non-
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pathogenic to humans.6 In soils, typical numbers of bacteria range from 108-1010 cells per gram.
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If one considers a homeowner on an acre lot, then the homeowner is surrounded by ~1018 native
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soil bacteria per acre furrow slice.
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One acre furrow slice (1 acre to a depth of 6”) ≃ 2 x 106 lbs soil ≃ 9 x 108 g soil
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Assuming 109 bacterial cells per g soil, it follows that there are
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109 x 9 x 108 cells per acre furrow slice
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≃ 1018 cells per acre furrow slice
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The numbers of intrinsic ARB within soils are also large. Such antibiotic resistance has been
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documented in pristine natural soils even when unimpacted by anthropogenic activities.24 Studies
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have shown that even pristine, undisturbed soils contain ARB,18 and that “environmental
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antibiotic resistance” has been a naturally-occurring factor in nature for more than 3 billion
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years.25,5 Culture-based estimates of ARB extracted from soils that were resistant to
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tetracycline, ciprofloxacin, cephalothin and ampicillin ranged from 106 to 107 colony forming
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units (CFU) per gram of soil.26 Likewise, Demaneche et al.27 demonstrated approximately 50-
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70% of cultured bacteria (tryptic soy agar) were ampicillin resistant (~104 CFU/g) from an
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undisturbed prairie soil. Durso et al.18 documented tetracycline- or cefotaxime-resistant
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heterotrophic counts ranging from 104 to 105 CFU/g in undisturbed prairie soil. Bacterial human
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pathogens have also been reported to enter a viable but non-culturable (VBNC) state.28 If the
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culture-based estimate of 106 to 107 resistant CFU per gram of soil is assumed, then a
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homeowner on an acre lot is surrounded by 1015 to 1016 antibiotic resistant bacteria that are
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resistant to one or more antibiotics. Many of the culturable bacteria are resistant to multiple
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antibiotics leading to the term: “The Soil Antibiotic Resistome.”29 Interestingly soil bacteria
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typically contain universal mechanisms of resistance to both natural and synthetic antibiotics.30
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In summary, every human is surrounded by extraordinarily large numbers of bacteria including
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vast numbers that can be resistant to any known antibiotic. Bacterial communities in natural
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environments unimpacted by anthropogenic activities also tend to be relatively stable with
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regards to taxonomic diversity and population density,31 but the influence of antibiotic inputs on
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the indigenous soil microbial community and the ARB community has shown variable effects.
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For example, a recent study in which streptomycin was applied to soil, demonstrated that the
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antibiotic did not influence the abundance nor diversity of the indigenous bacterial taxa.32 In
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contrast, others have reported localized or temporal shifts (
